Methods, reagents and cells for biosynthesizing compounds

ABSTRACT

This document describes biochemical pathways for producing 7-hydroxyheptanoate methyl ester and heptanoic acid heptyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase, and a monooxygenase, as well as recombinant hosts expressing one or more of such exogenous enzymes. 7-hydroxyheptanoate methyl esters and heptanoic acid heptyl esters can be enzymatically converted to pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/741,194, filed Jun. 16, 2015, which claims the benefit of U.S.Provisional Application Nos. 62/012,659, filed Jun. 16, 2014,62/012,666, filed Jun. 16, 2014, and 62/012,604, filed Jun. 16, 2014,the disclosure of each of which is incorporated herein by reference inits entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

An official copy of the sequence listing is submitted electronically viaEFS-Web as an ASCII formatted sequence listing with a file named“12444_0303-01000_SL.txt”, created on Jul. 5, 2017. Said ASCII copy is147,269 bytes in size. The sequence listing contained in this ASCIIformatted document is part of the specification and is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing7-hydroxyheptanoate methyl ester and heptanoic acid heptyl ester usingone or more isolated enzymes such as a fatty acid O-methyltransferase,an alcohol O-acetyltranferase, and a monooxygenase, and to recombinanthost cells expressing one or more such enzymes. This invention alsorelates to methods for enzymatically converting 7-hydroxyheptanoatemethyl ester and heptanoic acid heptyl ester to 7-hydroxyheptanoate and1,7-heptanediol using one or more enzymes such as an esterase, amonooxygenase, a demethylase, or an esterase, and recombinant hostsexpressing one or more such enzymes. In addition, this invention relatesto enzymatically converting 7-hydroxyheptanoate and/or 1,7-heptanediolto pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine or1,7-heptanediol (hereafter “C7 building blocks) and recombinant hostsproducing such C7 building blocks.

BACKGROUND

Nylons are polyamides which are generally synthesized by thecondensation polymerization of a diamine with a dicarboxylic acid.Similarly, Nylons may be produced by the condensation polymerization oflactams. A ubiquitous nylon is Nylon 6,6, which is produced bycondensation polymerization of hexamethylenediamine (HMD) and adipicacid. Nylon 6 can be produced by a ring opening polymerization ofcaprolactam (Anton & Baird, Polyamides Fibers, Encyclopedia of PolymerScience and Technology, 2001).

Nylon 7 and Nylon 7,7 represent novel polyamides with value-addedcharacteristics compared to Nylon 6 and Nylon 6,6. Nylon 7 is producedby polymerisation of 7-aminoheptanoic acid, whereas Nylon 7,7 isproduced by condensation polymerisation of pimelic acid andheptamethylenediamine. No economically viable petrochemical routes existto producing the monomers for Nylon 7 and Nylon 7,7.

Given no economically viable petrochemical monomer feedstocks,biotechnology offers an alternative approach via biocatalysis.Biocatalysis is the use of biological catalysts, such as enzymes, toperform biochemical transformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing one or more of pimelic acid,7-hydroxyheptanoate, 7-aminoheptanoate, heptamethylenediamine and1,7-heptanediol (hereafter “C7 building blocks”) wherein the methods arebiocatalyst based.

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes such C7 building blocks to the extracellular environment.Nevertheless, the metabolism of pimelic acid has been reported.

The dicarboxylic acid pimelic acid is converted efficiently as a carbonsource by a number of bacteria and yeasts via β-oxidation into centralmetabolites. β-oxidation of Coenzyme A (CoA) activated pimelate to CoAactivated 3-oxopimelate facilitates further catabolism via, for example,pathways associated with aromatic substrate degradation. The catabolismof 3-oxopimeloyl-CoA to acetyl-CoA and glutaryl-CoA by several bacteriahas been characterized comprehensively (Harwood and Parales, AnimalReview of Microbiology, 1996, 50:553-590).

The optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needfor expressing heterologous pathways in a host organism, directingcarbon flux towards C7 building blocks that serve as carbon sourcesrather than as biomass growth constituents, contradicts the optimalityprinciple. For example, transferring the 1-butanol pathway fromClostridium species into other production strains has often fallen shortby an order of magnitude compared to the production performance ofnative producers (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

The efficient synthesis of the seven carbon aliphatic backbone precursoris a key consideration in synthesizing one or more C7 building blocksprior to forming terminal functional groups, such as carboxyl, amine orhydroxyl groups, on the C7 aliphatic backbone.

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing a seven carbonchain aliphatic backbone precursor in which one or two functionalgroups, i.e., carboxyl, amine, or hydroxyl, can be formed, leading tothe synthesis of one or more of pimelic acid, 7-aminoheptanoate,7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol(hereafter “C7 building blocks). Pimelic acid and pimelate,7-hydroxyheptanoic acid and 7-hydroxyheptanoate, and 7-aminoheptanoicand 7-aminoheptanoate are used interchangeably herein to refer to therelevant compound in any of its neutral or ionized forms, including anysalt forms thereof. It is understood by those skilled in the art thatthe specific form will depend on pH.

One of skill in the art understands that compounds containing carboxylicacid groups (including, but not limited to, organic monoacids,hydroxyacids, aminoacids, and dicarboxylic acids) are formed orconverted to their ionic salt form when an acidic proton present in theparent compound either is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base. Acceptable organic bases include, but are not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases include, butare not limited to, aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. A salt ofthe present invention is isolated as a salt or converted to the freeacid by reducing the pH to below the pKa, through addition of acid ortreatment with an acidic ion exchange resin.

One of skill in the art understands that compounds containing aminegroups (including, but not limited to, organic amines, aminoacids, anddiamines) are formed or converted to their ionic salt form, for example,by addition of an acidic proton to the amine to form the ammonium salt,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids including, but not limited to, acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like. Acceptable inorganicbases include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like. A salt of the present invention is isolated as a salt orconverted to the free amine by raising the pH to above the pKb throughaddition of base or treatment with a basic ion exchange resin.

One of skill in the art understands that compounds containing both aminegroups and carboxylic acid groups (including, but not limited to,aminoacids) are formed or converted to their ionic salt form byeither 1) acid addition salts, formed with inorganic acids including,but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, and the like; or formed with organic acidsincluding, but not limited to, acetic acid, propionic acid, hexanoicacid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lacticacid, malonic acid, succinic acid, malic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like. Acceptable inorganicbases include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like, or 2) when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase. Acceptable organic bases include, but are not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases include, butare not limited to, aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. A salt canof the present invention is isolated as a salt or converted to the freeacid by reducing the pH to below the pKa through addition of acid ortreatment with an acidic ion exchange resin.

Pathways, metabolic engineering and cultivation strategies describedherein can rely on producing heptanoate methyl ester from heptanoateusing, for example, a fatty acid O-methyltransferase and producing7-hydroxyheptanoate methyl ester from heptanoate methyl ester using, forexample, a monooxygenase. 7-hydroxyheptanoate can be produced from7-hydroxyheptanoate methyl ester using, for example, a demethylase or anesterase.

Pathways, metabolic engineering and cultivation strategies describedherein also can rely on producing heptanoic acid heptyl ester using, forexample, an alcohol O-acetyltransferase and producing 7-hydroxyheptanoicacid heptyl ester, 7-hydroxyheptanoic acid 7-hydroxyheptyl ester and/orheptanoic acid 7-hydroxyheptyl ester from heptanoic acid heptyl esterusing, for example, a monooxygenase. 7-hydroxyheptanoate can be producedfrom 7-hydroxyheptanoic acid heptyl ester and/or 7-hydroxyheptanoic acid7-hydroxyheptyl ester using, for example, an esterase. 1,7-heptanediolcan be produced from heptanoic acid 7-hydroxyheptyl ester and/or7-hydroxyheptanoic acid 7-hydroxyheptyl ester using, for example, anesterase.

CoA-dependent elongation enzymes or homologs associated with the carbonstorage pathways from polyhydroxyalkanoate accumulating bacteria areuseful for producing precursor molecules. See, e.g., FIGS. 1-2.

In the face of the optimality principle, the inventors discoveredsurprisingly that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemical networkand cultivation strategies may be combined to efficiently produce one ormore C7 building blocks.

In some embodiments, the C7 aliphatic backbone for conversion to a C7building block can be formed from acetyl-CoA and propanoyl-CoA via twocycles of CoA-dependent carbon chain elongation using either NADH orNADPH dependent enzymes. See FIG. 1 and FIG. 2.

In some embodiments, an enzyme in the CoA-dependent carbon chainelongation pathway generating the C7 aliphatic backbone purposefullycontains irreversible enzymatic steps.

In some embodiments, the terminal carboxyl groups can be enzymaticallyformed using a thioesterase, an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a5-oxopentanoate dehydrogenase or a monooxygenase. See FIG. 3 and FIG. 4.

In some embodiments, the terminal amine groups can be enzymaticallyformed using a ω-transaminase or a deacetylase. See FIG. 5 and FIG. 6.

In some embodiments, the terminal hydroxyl group can be enzymaticallyformed using a monooxygenase, an esterase, or an alcohol dehydrogenase.See FIG. 3, FIG. 7 and FIG. 8. A monooxygenase (e.g., in combinationwith an oxidoreductase and/or ferredoxin) or an alcohol dehydrogenasecan enzymatically form a hydroxyl group. The monooxygenase can have atleast 70% sequence identity to any one of the amino acid sequences setforth in SEQ ID NOs: 14-16 or 28-29. An esterase can have at least 70%identity to the amino acid sequence set forth in SEQ ID NO: 27.

A ω-transaminase or a deacetylase can enzymatically form an amine group.The ω-transaminase can have at least 70/o sequence identity to any oneof the amino acid sequences set forth in SEQ ID NOs. 8-13.

A thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoatedehydrogenase, a 5-oxopenlanoate dehydrogenase, or a 6-oxohexanoatedehydrogenase can enzymatically form a terminal carboxyl group. Thethioesterase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1, and/or SEQ ID NO: 33-34.

A carboxylate reductase (e.g., in combination with a phosphopantetheinyltransferase) can form a terminal aldehyde group as an intermediate informing the product. The carboxylate reductase can have at least 70%sequence identity to any one of the amino acid sequences set forth inSEQ ID NOs: 2-7.

Any of the methods can be performed in a recombinant host byfermentation. The host can be subjected to a cultivation strategy underaerobic, anaerobic, or micro-aerobic cultivation conditions. The hostcan be cultured under conditions of nutrient limitation such asphosphate, oxygen or nitrogen limitation. The host can be retained usinga ceramic membrane to maintain a high cell density during fermentation.

In any of the methods, the host's tolerance to high concentrations of aC7 building block can be improved through continuous cultivation in aselective environment.

The principal carbon source fed to the fermentation can derive frombiological or non-biological feedstocks. In some embodiments, thebiological feedstock is, includes, or derives from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid and formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste.

In some embodiments, the non-biological feedstock is or derives fromnatural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatileresidue (NVR) or a caustic wash waste stream from cyclohexane oxidationprocesses, or a terephthalic acid/isophthalic acid mixture waste stream.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding a fatty acid O-methyltransferase anda monooxygenase, and produce 7-hydroxyheptanoate methyl ester. Such ahost further can include a demethylase or esterase and further produce7-hydroxyheptanoate. Such hosts further can include (i) a β-ketothiolaseor an acetyl-CoA carboxylase and a β-ketoacyl-[acp] synthase, (ii) a3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iii) anenoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoA reductase. The hostsalso further can include one or more of a thioesterase, an aldehydedehydrogenase, or a butanal dehydrogenase.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding an alcohol O-acetyltransferase andproduce heptanoic acid heptyl ester. Such a host further can include amonooxygenase and an esterase and further produce 7-hydroxyheptanoateand/or 1,7-heptanediol. Such hosts further can include (i) aβ-ketlothiolase or an acetyl-CoA carboxylase and a β-ketoacyl-[acp]synthase, (ii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoAreductase, (iii) an enoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoAreductase. The hosts also further can include one or more of athioesterase, a carboxylate reductase, an aldehyde dehydrogenase, abutanal or acetaldehyde dehydrogenase, or an alcohol dehydrogenase.

A recombinant host producing 7-hydroxyheptanoate further can include oneor more of a monooxygenase, an alcohol dehydrogenase, an aldehydedehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 6-oxohexanoatedehydrogenase, or a 7-oxoheptanoate dehydrogenase, the host furtherproducing pimelic acid or pimelate semialdehyde.

A recombinant host producing 7-hydroxyheptanoate further can include oneor more of a monooxygenase, a transaminase, a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyratedehydrogenase, and an alcohol dehydrogenase, wherein the host furtherproduces 7-aminoheptanoate.

A recombinant host producing 7-hydroxyheptanoate or 7-aminoheptanoatefurther can include one or more of a carboxylate reductase, aω-transaminase, a deacetylase, a N-acetyl transferase, or an alcoholdehydrogenate, the host further producing heptamethylenediamine.

A recombinant host producing 7-hydroxyheptanoate further can include acarboxylate reductase or an alcohol dehydrogenase, wherein the hostfurther produces 1,7-heptanediol.

The recombinant host can be a prokaryote, e.g., from the genusEscherichia such as Escherichia coli; from the genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the genus Corynebacteria such as Corynebacteriumglutamicum; from the genus Cupriavidus such as Cupriavidus necator orCupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonafluorecens, Pseudomonas putida or Pseudomonas oleavorans; from the genusDelftia acidovorans, from the genus Bacillus such as Bacillus subtillis;from the genes Lactobacillus such as Lactobacillus delbrueckii; from thegenus Lactococcus such as Lactococcus lactis or from the genusRhodococcus such as Rhodococcus equi.

The recombinant host can be a eukaryote, e.g., a eukaryote from thegenus Aspergillus such as Aspergillus niger; from the genusSaccharomyces such as Saccharomyces cerevisiae; from the genus Pichiasuch as Pichia pastoris; from the genus Yarrowia such as Yarrowialipolytica, from the genus Issatchenkia such as Issathenkia orientalis,from the genus Debaryomyces such as Debaryomyces hansenii, from thegenus Arxula such as Arxula adenoinivorans, or from the genusKluyveromryces such as Kluyveromyces lactis.

In some embodiments, the host's endogenous biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofacetyl-CoA and propanoyl-CoA, (2) create a cofactor, i.e. NADH or NADPH,imbalance that may be balanced via the formation of a C7 Building Block,(3) prevent degradation of central metabolites, central precursorsleading to and including C7 Building Blocks and (4) ensure efficientefflux from the cell.

Any of the recombinant hosts described herein further can include one ormore of the following attenuated enzymes: polyhydroxyalkanoate synthase,an acetyl-CoA thioesterase, a propanoyl-CoA thioesterase, amethylcitrate synthase, an acetyl-CoA specific β-ketothiolase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fitmarate oxidoreductase, a 2-oxoaciddecarboxylase producing isobutanol, an alcohol dehydrogenase formingethanol, a triose phosphate isomerase, a pyruvate decarboxylase, aglucose-6-phosphate isomerase, a transhydrogenase dissipating thecofactor imbalance, a glutamate dehydrogenase specific for the co-factorfor which an imbalance is created, a NADH/NADPH-utilizing glutamatedehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenaseaccepting C7 building blocks and central precursors as substrates; aglutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: an acetyl-CoA synthetase, a6-phosphogluconate dehydrogenase; a transketolase; a feedback resistantthreonine deamninase; a puridine nucleotide transhydrogenase; a formatedehydrogenase; a glyceraldehyde-3P-dehydrogenae; a malic enzyme; aglucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; apropionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamatedehydrogenase; a L-glutamine synthetase; a lysine transporter; adicarboxylate transporter; and/or a multidrug transporter.

This document also features methods of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester, the method including enzymaticallyconverting a C₄₋₉ carboxylic acid to a (C₃₋₈ alkyl)-C(═O)OCH₃ ester; andenzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

In some embodiments, the C₄₋₉ carboxylic acid can be enzymaticallyconverted to the (C₃₋₉ alkyl)-C(═O)OCH₃ ester using a polypeptide havingfatty acid O-methyltransferase activity. In some embodiments, thepolypeptide having fatty acid O-methyltransferase activity can have atleast 70% sequence identity to an amino acid sequence set forth in SEQID NO:23, SEQ ID NO:24, and/or SEQ ID NO:25.

In some embodiments, the (C₃₋₈ alkyl)-C(═O)OCH₃ ester can beenzymatically converted to the (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester usinga polypeptide having monooxygenase activity. In some embodiments, themonooxygenase is classified under EC 1.14.14.- or EC 1.14.15.-. In someembodiments, the monooxygenase can have at least 70% sequence identityto an amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO:15, SEQID NO: 16, SEQ ID NO:28 and/or SEQ ID NO:29.

In some embodiments, the C₄₋₉ carboxylic acid can be enzymaticallyproduced from a C₄₋₉ alkanoyl-CoA. In some embodiments, a polypeptidehaving thioesterase activity can enzymatically produce the C₄₋₉carboxylic acid from the C₄₋₉ alkanoyl-CoA.

In some embodiments, the thioesterase can have at least 70% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1 and/or SEQID NO: 33-34. In some embodiments, a polypeptide having butanaldehydrogenase activity and a polypeptide having aldehyde dehydrogenaseactivity enzymatically produce the C₄₋₉ carboxylic acid from C₄₋₉alkanoyl-CoA.

This document also features methods of producing one or morehydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters. The methodincludes enzymatically converting a C₄₋₉ alkanoyl-CoA to a (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester; and enzymatically converting the (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester.

In some embodiments, the C₄₋₉ alkanoyl-CoA can be enzymaticallyconverted to the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having alcohol O-acetyltransferase activity. In someembodiments, the alcohol O-acetyltransferase can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester can beenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having monooxygenase activity. In some embodiments, thepolypeptide having monooxygenase activity can be classified under EC1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further can include enzymaticallyconverting the (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or(C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester to a C₄₋₉hydroxyalkanoate. In some embodiments, a polypeptide having esteraseactivity enzymatically converts the (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester tothe C₄₋₉ hydroxyalkanoate.

In one aspect, this document features a method for producing abioderived seven carbon compound. The method for producing a bioderivedseven carbon compound can include culturing or growing a recombinanthost as described herein under conditions and for a sufficient period oftime to produce the bioderived seven carbon compound, wherein,optionally, the bioderived seven carbon compound is selected from thegroup consisting of pimelic acid, pimelate semialdehyde,7-aminoheptanoate acid, 7-hydroxyheptanoate, heptamethylenediamine,1,7-heptanediol, and combinations thereof.

In one aspect, this document features composition comprising abioderived seven carbon compound as described herein and a compoundother than the bioderived seven carbon compound, wherein the bioderivedseven carbon compound is selected from the group consisting of pimelicacid, pimelate semialdehyde, 7-aminoheptanoate acid,7-hydroxyheptanoate, heptamethylenediamine, 1,7-heptanediol, andcombinations thereof. For example, the bioderived seven carbon compoundis a cellular portion of a host cell or an organism.

This document also features a biobased polymer comprising the bioderivedpimelic acid, pimelate semialdehyde, 7-aminoheptanoate acid,7-hydroxyheptanoate, heptamethylenediamine, 1,7-heptanediol, andcombinations thereof.

This document also features a biobased resin comprising the bioderivedpimelic acid, pimelate semialdehyde, 7-aminoheptanoate acid,7-hydroxyheptanoate, heptamethylenediamine, 1,7-heptanediol, andcombinations thereof as well as a molded product obtained by molding abiobased resin.

In another aspect, this document features a process for producing abiobased polymer that includes chemically reacting the bioderivedpimelic acid, pimelate semialdehyde, 7-aminoheptanoate acid,7-hydroxyheptanoate, heptamethylenediamine, 1,7-heptanediol, andcombinations thereof with itself or another compound in a polymerproducing reaction.

In another aspect, this document features a process for producing abiobased resin that includes chemically reacting the bioderived pimelicacid, pimelate semialdehyde, 7-aminoheptanoate acid,7-hydroxyheptanoate, heptamethylenediamine, 1,7-heptanediol, andcombinations thereof with itself or another compound in a resinproducing reaction.

In another aspect, this document provides a bio-derived product,bio-based product or fermentation-derived product, wherein said productcomprises:

(i) a composition comprising at least one bio-derived, bio-based orfermentation-derived compound provided herein or in any one of FIGS.1-9, or any combination thereof;

(ii) a bio-derived, bio-based or fermentation-derived polymer comprisingthe bio-derived, bio-based or fermentation-derived composition orcompound of (i), or any combination thereof;

(iii) a bio-derived, bio-based or fermentation-derived resin comprisingthe bio-derived, bio-based or fermentation-derived compound orbio-derived, bio-based or fermentation-derived composition of (i) or anycombination thereof or the bio-derived, bio-based orfermentation-derived polymer of (ii) or any combination thereof; (iv) amolded substance obtained by molding the bio-derived, bio-based orfermentation-derived polymer of (ii) or the bio-derived, bio-based orfermentation-derived resin of (iii), or any combination thereof;

(v) a bio-derived, bio-based or fermentation-derived formulationcomprising the bio-derived, bio-based or fermentation-derivedcomposition of (i), bio-derived, bio-based or fermentation-derivedcompound of (i), bio-derived, bio-based or fermentation-derived polymerof (ii), bio-derived, bio-based or fermentation-derived resin of (iii),or bio-derived, bio-based or fermentation-derived molded substance of(iv), or any combination thereof; or

(vi) a bio-derived, bio-based or fermentation-derived semi-solid or anon-semi-solid stream, comprising the bio-derived, bio-based orfermentation-derived composition of (i), bio-derived, bio-based orfermentation-derived compound of (i), bio-derived, bio-based orfermentation-derived polymer of (ii), bio-derived, bio-based orfermentation-derived resin of (iii), bio-derived, bio-based orfermentation-derived formulation of (v), or bio-derived, bio-based orfermentation-derived molded substance of (iv), or any combinationthereof.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having (i) fatty acidO-methyltransferase activity or alcohol O-acetyltransferase activity,(ii) monooxygenase activity, and (iii) esterase or demethylase activity.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having fatty acidO-methyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the biochemical network enzymatically produces7-hydroxyheptanoate methyl ester. The biochemical network can furtherinclude a polypeptide having demethylase activity or a polypeptidehaving esterase activity, wherein the polypeptide having demethylaseactivity or a polypeptide having esterase activity enzymatically produce7-hydroxyheptanoate.

The biochemical network can further include at least one exogenousnucleic acid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity, wherein the polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity enzymatically produce C7 precursor molecules such asheptanoyl-CoA.

The biochemical network can further one or more of an exogenouspolypeptide having thioesterase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having butanal dehydrogenaseactivity, wherein the polypeptide having thioesterase activity, apolypeptide having aldehyde dehydrogenase activity, or a polypeptidehaving butanal dehydrogenase activity enzymatically produce heptanoateas a C7 precursor molecule.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having alcoholO-acetyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the biochemical network produces heptanoic acid heptylester. The biochemical network can further include an esterase, whereinthe esterase enzymatically converts heptanoic acid heptyl ester to7-hydroxyheptanoate and/or 1,7-heptanediol.

The biochemical network can further include at least one exogenousnucleic acid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity, wherein the polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity enzymatically produce C7 precursor molecules such asheptanoyl-CoA. The biochemical network can further include one or moreof an exogenous a polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activity,wherein the polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activityenzymatically produce heptanol as a C7 precursor molecule.

A biochemical network producing 7-hydroxyheptanoate can further includeone or more of a polypeptide having monooxygenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingaldehyde dehydrogenase activity, a polypeptide having 7-oxohexanoatedehydrogenase activity, a polypeptide having 6-oxohexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity or a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, wherein the polypeptide having monooxygenaseactivity, a polypeptide having alcohol dehydrogenase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity enzymatically convert7-hydroxyheptanoate to pimelic acid or pimelate semialdehyde.

A biochemical network producing 7-hydroxyheptanoate can further includeone or more of a polypeptide having ω-transaminase activity, apolypeptide having 6-hydroxyhexanoate dehydrogenase activity, apolypeptide having 5-hydroxybutanoate dehydrogenase activity, apolypeptide having 4-hydroxybutyrate dehydrogenase activity and apolypeptide having alcohol dehydrogenase activity, wherein thepolypeptide having ω-transaminase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity and a polypeptide havingalcohol dehydrogenase activity enzymatically convert 7-hydroxyheptanoateto 7-aminoheptanoate.

A biochemical network producing 7-aminoheptanoate, 7-hydroxyheptanoate,pimelate semialdehyde, or 1,7-heptanediol can further include one ormore of a polypeptide having carboxylate reductase activity, apolypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, a polypeptide having N-acetyl transferaseactivity, or a polypeptide having alcohol dehydrogenase activity,wherein the a polypeptide having carboxylate reductase activity, apolypeptide having co-transaminase activity, a polypeptide havingdeacetylase activity, a polypeptide having N-acetyl transferaseactivity, or a polypeptide having alcohol dehydrogenase activity,enzymatically convert 7-aminoheptanoate, 7-hydroxyheptanoate, pimelatesemialdehyde, or 1,7-heptanediol to heptamethylenediamine.

A biochemical network producing 7-hydroxyheptanoate can further includeone or more of a polypeptide having carboxylate reductase activity and apolypeptide having alcohol dehydrogenase activity, wherein thepolypeptide having carboxylate reductase activity and a polypeptidehaving alcohol dehydrogenase activity enzymatically convert7-hydroxyheptanoate to 1,7-heptanediol.

Also, described herein is a means for obtaining 7-hydroxyheptanoateusing (i) a polypeptide having fatty acid O-methyltransferase activityand a polypeptide having monooxygenase activity and (ii) a polypeptidehaving demethylase activity or a polypeptide having esterase activity.The means can further include means for converting 7-hydroxyhexanoate toat least one of pimelic acid, 7-aminoheptanoic acid,heptamethylenediamine, 7-hydroxyheptanoate, and 1,7-heptanediol. Themeans can include a polypeptide having aldehyde dehydrogenase activity,a polypeptide having 7-oxohexanoate dehydrogenase activity, apolypeptide having 6-oxohexanoate dehydrogenase activity, a polypeptidehaving 5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity.

Also, described herein is a means for obtaining 7-hydroxyheptanoateusing (i) a polypeptide having alcohol O-acetyltransferase and apolypeptide having monooxygenase activity and (ii) a polypeptide havingdemethylase activity or a polypeptide having esterase activity. Themeans can further include means for converting 7-hydroxyheptanoate to atleast one of pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine,7-hydroxyheptanoic acid, and 1,7-heptanediol. The means can include apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity.

Also described herein is (i) a step for obtaining 7-hydroxyheptanoateusing a polypeptide having alcohol O-acetyltransferase, a polypeptidehaving monooxygenase activity, and a polypeptide having demethylaseactivity or a polypeptide having esterase activity, and (ii) a step forobtaining pimelic acid, 7-aminoheptanoate, pimelate semialdehyde1,7-heptanediol, or heptamethylenediamine using a polypeptide havingcarboxylate reductase activity, a polypeptide having alcoholdehydrogenase activity, a polypeptide having ω-transaminase activity, apolypeptide having deacetylase activity, a polypeptide having N-acetyltransferase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxybutanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, a polypeptide having aldehyde dehydrogenaseactivity, a polypeptide having 7-oxohexanoate dehydrogenase activity, apolypeptide having 6-oxohexanoate dehydrogenase activity, a polypeptidehaving 5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity,

In another aspect, this document features a composition comprising7-hydroxyheptanoate and a polypeptide having alcoholO-acetyltransferase, a polypeptide having monooxygenase activity, and apolypeptide having demethylase activity or a polypeptide having esteraseactivity complex. The composition can be cellular. The composition canfurther include a polypeptide having carboxylate reductase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingco-transaminase activity, a polypeptide having deacetylase activity, apolypeptide having N-acetyl transferase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, a polypeptide having 7-oxohexanoatedehydrogenase activity, a polypeptide having 6-oxohexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity or a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, and at least one of pimelic acid,7-aminoheptanoic acid, heptamethylenediamine, 7-hydroxyheptanoic acid,and 1,7-heptanediol. The composition can be cellular.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from of theabove types of host cells and used in a purified or semi-purified form.Extracted enzymes can optionally be immobilized to the floors and/orwalls of appropriate reaction vessels. Moreover, such extracts includelysates (e.g. cell lysates) that can be used as sources of relevantenzymes. In the methods provided by the document, all the steps can beperformed in cells (e.g., host cells), all the steps can be performedusing extracted enzymes, or some of the steps can be performed in cellsand others can be performed using extracted enzymes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein including GenBank and NCBI submissions with accessionnumbers are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and the drawings, and from the claims. The word “comprising”in the claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of exemplary biochemical pathways leading toheptanoyl-CoA using NADH-dependent enzymes and acetyl-CoA andpropanoyl-CoA as central metabolites.

FIG. 2 is a schematic of exemplary biochemical pathways leading toheptanoyl-CoA using NADPH-dependent enzymes and acetyl-CoA andpropanoyl-CoA as central metabolites.

FIG. 3 is a schematic of exemplary biochemical pathways leading toheptanoate and heptanol using heptanoyl-CoA as a central precursor.

FIG. 4 is a schematic of exemplary biochemical pathways leading topimelic acid using 7-hydroxyheptanoate as a central precursor.

FIG. 5 is a schematic of an exemplary biochemical pathway leading to7-aminoheptanoate using 7-hydroxyheptanoate as a central precursor.

FIG. 6 is a schematic of exemplary biochemical pathways leading toheptamethylenediamine using 7-aminoheptanoate, 7-hydroxyheptanoate,pimelate semialdehyde, or 1,7-heptanediol as a central precursor.

FIG. 7 is a schematic of exemplary biochemical pathways leading to7-hydroxyheptanoate via ester intermediates using heptanoate orheptanoyl-CoA. FIG. 7 also contains an exemplary biochemical pathwayleading to 7-hydroxyheptanoate using 1,7-heptanediol as a centralprecursor.

FIG. 8 is a schematic of exemplary biochemical pathways leading to 1,7heptanediol using 7-hydroxyheptanoate or heptanoyl-CoA as a centralprecursor.

FIG. 9 is a schematic of exemplary biochemical pathways leading topropanoyl-CoA from central metabolites.

FIGS. 10A-10L contain the amino acid sequences of an Escherichia colithioesterase encoded by tesB (see GenBank Accession No. AAA24665.1, SEQID NO: 1), a Mycobacterium marinum carboxylate reductase (see GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (see Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis carboxylatereductase (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), aMycobacterium massiliense carboxylate reductase (see Genbank AccessionNo. EIV11143.1, SEQ ID NO: 6), a Segniliparus rotundus carboxylatereductase (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), aChromobacterium violaceurm ω-transaminase (see Genbank Accession No.AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa a)transaminase (seeGenbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringaeω-transaminase (see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), aRhodobacter sphaeroides ω-transaminase (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli ω-transaminase (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialisω-transaminase (See Genbank Accession No. AEA39183.1, SEQ ID NO: 13); aPolaromonas sp. JS666 monooxygenase (see Genbank Accession No.ABE47160.1, SEQ ID NO: 14), a Mycobacterium sp. HX7V-1500 monooxygenase(see Genbank Accession No. CAH04396.1, SEQ ID NO: 15), a Mycobacteriumaustroafricanum monooxygenase (see Genbank Accession No. ACJ06772.1, SEQID NO: 16), a Polaromonas sp. JS666 oxidoreductase (see GenbankAccession No. ABE47159.1, SEQ ID NO: 17), a Mycobacterium sp. HX7-1500oxidoreductase (see Genbank Accession No. CAH04397.1, SEQ ID NO: 18), aPolaromonas sp. JS666 ferredoxin (see Genbank Accession No. ABE47158.1,SEQ ID NO: 19), a Mycobacterium sp. HXN-1500 ferredoxin (see GenbankAccession No. CAH04398.1, SEQ ID NO:20), Bacillus subtilisphosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1,SEQ ID NO:21), a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase(see Genbank Accession No. ABI83656.1, SEQ ID NO:22), a Mycobacteriummarinum fatty acid O-methyltransferase (GenBank Accession No.ACC41782.1; SEQ ID NO:23), a Mycobacterium smegmatis str. MC2 fatty acidO-methyltransferase (GenBank Accession No. ABK73223.1; SEQ ID NO:24), aPseudomonas putida fatty acid O-methyltransferase (GenBank Accession No.CAA39234.1; SEQ ID NO:25), a Saccharomyces cerevisiae alcoholO-acetyltransferase (Genbank Accession No: CAA85138.1, SEQ ID NO: 26), aPseudomonas fluorescens carboxylesterase (Genbank Accession No.AAC60471.2, SEQ ID NO: 27), a Pseudomonas putida alkane 1-monooxygenase(Genbank Accession No. CAB51047.1, SEQ ID NO: 28), a Candida maltosecytochrome P450 (Genbank Accession No: BAA00371.1, SEQ ID NOs: 29), aSalmonella enterica subsp. enterica serovar Typhinmurium butanaldehydrogenase (GenBank Accession No. AAD39015, SEQ ID NO:30), aSphingomonas paucimobilis demethylase (GenBank Accession No. BAD61059.1and GenBank Accession No. BAC79257.1, SEQ ID NOs: 31 and 32,respectively), a Lactobacillus brevis thioesterase (GenBank AccessionNo. ABJ63754.1, SEQ ID NO:33), and a Lactobacillus plantarumthioesterase (GenBank Accession No. CCC78182.1, SEQ ID NO:34).

FIG. 11 is a bar graph of the change in peak area after 24 hours for7-hydroxyheptanoate as determined via liquid chromatograph (LC)-massspectrometry (MS), as a measure of the monooxygenase activity of SEQ IDNOs: 14-16 (GenBank Accession Nos: ABE47160.1, CAH04396.1, andACJ06772.1, respectively) for converting heptanoate to7-hydroxyheptanoate relative to the empty vector control.

FIG. 12 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH andactivity of six carboxylate reductase preparations in enzyme onlycontrols (no substrate).

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof two carboxylate reductase preparations for converting pimelate topimelate semialdehyde relative to the empty vector control.

FIG. 14 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof six carboxylate reductase preparations for converting7-hydroxyheptanoate to 7-hydroxyheptanal relative to the empty vectorcontrol.

FIG. 15 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof three carboxylate reductase preparations for convertingN7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal relative to theempty vector control.

FIG. 16 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofa carboxylate reductase preparation for converting pimelate semialdehydeto heptanedial relative to the empty vector control.

FIG. 17 is a bar graph summarizing the percent conversion of pyruvate toL-alanine (mol/mol) as a measure of the (ω-transaminase activity of theenzyme only controls (no substrate).

FIG. 18 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the (ω-transaminaseactivity of four artransaminase preparations for converting7-aminoheptanoate to pimelate semialdehyde relative to the empty vectorcontrol.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity of three ω-transaminase preparations for converting pimelatesemialdehyde to 7-aminoheptanoate relative to the empty vector control.

FIG. 20 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of six ω-transaminase preparations for convertingheptamethylenediamine to 7-aminoheptanal relative to the empty vectorcontrol.

FIG. 21 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of six ω-transaminase preparations for convertingN7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal relative tothe empty vector control.

FIG. 22 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of three ω-transaminase preparations for converting7-aminoheptanol to 7-oxoheptanol relative to the empty vector control.

FIG. 23 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases of six carboxylate reductase preparations forconverting heptanoic acid to heptanal relative to the empty vectorcontrol.

DETAILED DESCRIPTION

This document provides enzymes, non-natural pathways, cultivationstrategies, feedstocks, central precursors, host microorganisms andattenuations to the host's biochemical network, which generate a sevencarbon chain aliphatic backbone (which can be bound to a coenzyme Amoiety) from central metabolites in which one or two terminal functionalgroups may be formed leading to the synthesis of one or more of pimelicacid, 7-hydroxyheptanoate, 7-aminoheptanoate, heptamethylenediamine or1,7-heptanediol (referred to as “C7 building blocks” herein). As usedherein, the term “central precursor” is used to denote any metabolite inany metabolic pathway shown herein leading to the synthesis of a C7building block. The term “central metabolite” is used herein to denote ametabolite that is produced in all microorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C7 building blocks orcentral precursors thereof can be produced. In an endogenous pathway,the host microorganism naturally expresses all of the enzymes catalyzingthe reactions within the pathway. A host microorganism containing anengineered pathway does not naturally express all of the enzymescatalyzing the reactions within the pathway but has been engineered suchthat all of the enzymes within the pathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following polypeptides may be expressed in thehost in addition to a polypeptide having fatty acid O-methyltransferaseactivity or a polypeptide having alcohol O-acetyltransferase activity:polypeptide having a monooxygenase activity, a polypeptide havingesterase activity, polypeptide having demethylase activity, apolypeptide having β-ketothiolase activity, a polypeptide havingacetyl-CoA carboxylase activity, a polypeptide having β-ketoacyl-[acp]synthase activity, a polypeptide having 3-hydroxyacyl-CoA dehydrogenaseactivity, a polypeptide having 3-oxoacyl-CoA reductase activity, apolypeptide having enoyl-CoA hydratase activity, a polypeptide havingtrans-2-enoyl-CoA reductase activity, a polypeptide having thioesteraseactivity, a polypeptide having aldehyde dehydrogenase activity, apolypeptide having butanal dehydrogenase activity, a polypeptide havingmonooxygenase activity in, for example, the CYP4F3B family, apolypeptide having alcohol dehydrogenase activity, a polypeptide having5-oxopenlanoate dehydrogenase activity, a polypeptide having6-oxohexanoale dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide havingω-transaminase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, a polypeptide having carboxylate reductaseactivity, a polypeptide having deacetylase activity, or a polypeptidehaving N-acetyl transferase activity. In recombinant hosts expressing apolypeptide having carboxylate reductase activity, a polypeptide havingphosphopantetheinyl transferase activity also can be expressed as itenhances activity of the carboxylate reductase. In recombinant hostsexpressing a polypeptide having monooxygenase activity, an electrontransfer chain protein such as a polypeptide having oxidoreductaseactivity and/or polypeptide having ferredoxin polypeptide activity alsocan be expressed.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having (i) fatty acidO-methyltransferase activity or alcohol O-acetyltransferase activity,(ii) polypeptide having monooxygenase activity, and (iii) a polypeptidehaving esterase activity or a polypeptide having demethylase activity.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having fatty acidO-methyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the host produces 7-hydroxyheptanoate methyl ester.Such a host further can include a polypeptide having demethylaseactivity or polypeptide having esterase activity and further produce7-hydroxyheptanoate. In some embodiments, the recombinant host also caninclude at least one exogenous nucleic acid encoding a polypeptidehaving β-ketothiolase activity or a polypeptide having acetyl-CoAcarboxylase activity and a polypeptide having β-ketoacyl-[acp] synthaseactivity, a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activityor a polypeptide having 3-oxoacyl-CoA reductase activity, a polypeptidehaving enoyl-CoA hydratase activity, and a polypeptide havingtrans-2-enoyl-CoA reductase activity to produce C7 precursor moleculessuch as heptanoyl-CoA. Such a host further can include one or more of(e.g., two or three of) an exogenous polypeptide having thioesteraseactivity, a polypeptide having aldehyde dehydrogenase activity, or apolypeptide having butanal dehydrogenase activity, and produceheptanoate as a C7 precursor molecule.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having alcoholO-acetyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the host produces heptanoic acid heptyl ester. Such ahost further can include a polypeptide having esterase activity andfurther produce 7-hydroxyheptanoate and/or 1,7-heptanediol. In someembodiments, the recombinant host also can include at least oneexogenous nucleic acid encoding a polypeptide having β-ketothiolaseactivity or a polypeptide having acetyl-CoA carboxylase activity and apolypeptide having β-ketoacyl-[acp] synthase activity, a polypeptidehaving 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to produce C7 precursor molecules such as heptanoyl-CoA. Such ahost further can include one or more of (e.g., two or three of) anexogenous polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activity andproduce heptanol as a C7 precursor molecule.

A recombinant host producing 7-hydroxyheptanoate further can include oneor more of a polypeptide having monooxygenase activity (e.g., in theCYP4F3B family), a polypeptide having alcohol dehydrogenase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having7-oxoheptanoate dehydrogenase activity, and produce pimelic acid. Forexample, a recombinant host further can include a monooxygenase andproduce pimelic acid. As another example, a recombinant host further caninclude (i) a polypeptide having alcohol dehydrogenase activity, apolypeptide having 6-hydroxyhexanoate dehydrogenase activity, apolypeptide having 5-hydroxypentanoate dehydrogenase activity, or apolypeptide having 4-hydroxybutyrate dehydrogenase activity or (ii) apolypeptide having aldehyde dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, or a polypeptide having7-oxoheptanoate dehydrogenase activity, and produce pimelic acid.

A recombinant host producing 7-hydroxyheptanoate further can include oneor more of a polypeptide having transaminase activity, a polypeptidehaving 6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity and a polypeptide havingalcohol dehydrogenase activity, and produce 7-aminoheptanoate. Forexample, a recombinant host producing 7-hydroxyheptanoate further caninclude a polypeptide having ω-transaminase activity and either apolypeptide having 6-hydroxyhexanoate dehydrogenase activity orpolypeptide having alcohol dehydrogenase activity.

A recombinant host producing 7-aminoheptanoate, 7-hydroxyheptanoate,pimelate semialdehyde or 1,7-heptanediol further can include one or moreof a polypeptide having carboxylate reductase activity, a polypeptidehaving ω-transaminase activity, a polypeptide having deacetylaseactivity, a polypeptide having N-acetyl transferase activity, or apolypeptide having alcohol dehydrogenase activity, and produceheptamethylenediamine. In some embodiments, a recombinant host furthercan include each of a polypeptide having carboxylate reductase activity,a polypeptide having ω-transaminase, a polypeptide having deacetylaseactivity, and a polypeptide having N-acetyl transferase activity. Insome embodiments, a recombinant host further can include a polypeptidehaving carboxylate reductase activity and a polypeptide havingω-transaminase activity. In some embodiments, a recombinant host furthercan include a polypeptide having carboxylate reductase activity, apolypeptide having ω-transaminase activity, and a polypeptide havingalcohol dehydrogenase activity. In the embodiments in which therecombinant host produces 7-aminoheptanoate, an additional polypeptidehaving ω-transaminase activity may not be necessary to produceheptamethylenediamine. In some embodiments, the host includes a secondexogenous polypeptide having ω-transaminase activity that differs fromthe first exogenous polypeptide having ω-transaminase activity.

A recombinant host producing 7-hydroxyheptanoic acid further can includeone or more of a polypeptide having carboxylate reductase activity and apolypeptide having alcohol dehydrogenase activity, and produce1,7-heptanediol.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genus, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL. In recombinant hosts containing an exogenous enzyme, the hostscontain an exogenous nucleic acid encoding the enzyme.

Any of the enzymes described herein that can be used for production ofone or more C7 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of thecorresponding wild-type enzyme. It will be appreciated that the sequenceidentity can be determined on the basis of the mature enzyme (e.g., withany signal sequence removed) or on the basis of the immature enzyme(e.g., with any signal sequence included). It also will be appreciatedthat the initial methionine residue may or may not be present on any ofthe enzyme sequences described herein.

For example, a polypeptide having thioesterase activity described hereincan have at least 70% sequence identity (homology) (e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) tothe amino acid sequence of an Escherichia coli thioesterase encoded bytesB (see GenBank Accession No. AAA24665.1, SEQ ID NO: 1), to the aminoacid sequence of a Lactobacillus brevis thioesterase (GenBank AccessionNo. ABJ63754.1, SEQ ID NO:33) or a Lactobacillus plantarum esterase(GenBank Accession No. CCC78182.1, SEQ ID NO:34). See FIG. 10A and FIG.10L.

For example, a polypeptide having carboxylate reductase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum(see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacteriumsmegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), aSegniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO:4), a Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1, SEQID NO: 5), a Mycobacterium massiliense (see Genbank Accession No.EIV1143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see GenbankAccession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. See,FIGS. 10A-10F.

For example, a polypeptide having ω-transaminase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 9), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:10), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1,SEQ ID NO: 11), an Escherichia coli (see Genbank Accession No.AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank AccessionNo. AEA39183.1, SEQ ID NO: 13) ω-transaminase. Some of thesepolypeptides having ω-transaminase activity are polypeptides havingdiamine ω-transaminase activity. See, FIGS. 10F-10H.

For example, a polypeptide having monooxygenase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Polaromonas sp. JS666monooxygenase (see Genbank Accession No. ABE47160.1, SEQ ID NO: 14), aMycobacterium sp. HXN-1500 monooxygenase (see Genbank Accession No.CAH04396.1, SEQ ID NO: 15), or a Mycobacterium austroafricanummonooxygenase (See Genbank Accession No. ACJ06772.1, SEQ ID NO: 16).See, FIG. 10H.

For example, a polypeptide having oxidoreductase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Polaromonas sp. JS666oxidoreductase (see Genbank Accession No. ABE47159.1, SEQ ID NO: 17) ora Mycobacterium sp. HXN-1500 oxidoreductase (see Genbank Accession No.CAH04397.1, SEQ ID NO:18). See, FIG. 10I.

For example, a polypeptide having ferredoxin activity described hereincan have at least 70% sequence identity (homology) (e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) tothe amino acid sequence of a Polaromonas sp. JS666 ferredoxin (seeGenbank Accession No. ABE47158.1, SEQ ID NO: 19) or a Mycobacterium sp.HXN-1500 ferredoxin (see Genbank Accession No. CAH04398.1, SEQ IDNO:20). See, FIG. 10I.

For example, a polypeptide having phosphopantetheinyl transferaseactivity described herein can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillussubtilis phosphopantetheinyl transferase (see Genbank Accession No.CAA44858.1, SEQ ID NO:21) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:22). See FIG. 10I.

For example, a polypeptide having fatty acid O-methyltransferaseactivity described herein can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marimim (see GenBank Accession No. ACC41782.1, SEQ ID NO:23), a Mycobacterium smegmatis (see GenBank Accession No. ABK73223.1,SEQ ID NO: 24), or a Pseudomonas putida (see GenBank Accession No.CAA39234.1, SEQ ID NO: 25) methyltransferase. See FIG. 10I and FIG. 10J.

For example, a polypeptide having alcohol O-acetyltransferase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Saccharomycescerevisiae (see GenBank Accession No. CAA85138.1, SEQ ID NO: 26) alcoholO-acetytransferase. See FIG. 10J.

For example, a polypeptide having esterase activity described herein canhave at least 70% sequence identity (homology) (e.g., at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to theamino acid sequence of a Pseudomonas fluorescens (see GenBank AccessionNo. AAC60471.2, SEQ ID NO: 27) esterase. See FIG. 10J.

For example, a polypeptide having alkane 1-monooxygenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas putidaalkane 1-monooxygenase (see Genbank Accession No. CAB51047.1, SEQ ID NO:28).

For example, a polypeptide having cytochrome P450 monooxygenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Candida maltosecytochrome P450 (see Genbank Accession No: BAA00371.1, SEQ ID NOs: 29).

For example, a polypeptide having butanal dehydrogenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Salmonella entericasubsp. enterica serovar Typhimurium butanal dehydrogenase (see GenBankAccession No. AAD39015, SEQ ID NO:30).

For example, a polypeptide having syringate O-demethylase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Sphingomonaspaucimobilis demethylase (see, GenBank Accession No. BAD61059.1 andGenBank Accession No. BAC79257.1, SEQ ID NOs: 31 and 32, respectively).

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\B12seq -i c:seq1.txt -j c:\seq2.txt-p blastp -o c:\output.txt. If the two compared sequences share homology(identity), then the designated output file will present those regionsof homology as aligned sequences. If the two compared sequences do notshare homology (identity), then the designated output file will notpresent aligned sequences. Similar procedures can be following fornucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that is shorterthan the full-length immature protein protein and has at least 25%(e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%;99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more,two or more, three or more, four or more, five or more, or six or more)of the enzymes of the pathways described herein. Thus, a pathway withinan engineered host can include all exogenous enzymes, or can includeboth endogenous and exogenous enzymes. Endogenous genes of theengineered hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Engineeredhosts can be referred to as recombinant hosts or recombinant host cells.As described herein recombinant hosts can include nucleic acids encodingone or more of a polypeptide having fatty acid O-methyltransferaseactivity, a polypeptide having alcohol O-acetyltransferase activity, apolypeptide having dehydrogenase activity, a polypeptide havingβ-ketothiolase activity, a polypeptide having β-ketoacyl-[acp] synthaseactivity, a polypeptide having carboxylase activity, a polypeptidehaving reductase activity, a polypeptide having hydratase activity, apolypeptide having thioesterase activity, a polypeptide havingmonooxygenase activity, a polypeptide having demethylase activity, apolypeptide having esterase activity, or a polypeptide havingtransaminase activity as described herein.

In addition, the production of one or more C7 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

Biosynthetic Methods

The present document provides methods of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester. As used herein, the term (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester refers to a compound having the followingformula:

As used herein, the term “C₃₋₈ hydroxyalkyl” refers to a saturatedhydrocarbon group that may be straight-chain or branched, and issubstituted by at least one hydroxyl (i.e., hydroxy or OH) group. Insome embodiments, the C₃₋₈ hydroxyalkyl refers to refers to a saturatedhydrocarbon group that may be straight-chain or branched, and issubstituted by at least one terminal hydroxyl (OH) group. In someembodiments, the alkyl group contains 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8 carbon atoms. In someembodiments, the C₃₋₈ hydroxyalkyl is a group of the following formula:

In some embodiments, the method comprises:

a) enzymatically converting a C₄₋₉ carboxylic acid to a (C₃₋₈alkyl)-C(═O)OCH₃ ester; and

b) enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

As used herein, the term “C₄₋₉ carboxylic acid” refers to a compoundhaving the formula R—C(═O)OH, wherein R is a refers to a saturatedhydrocarbon group (i.e., an alkyl group) that may be straight-chain orbranched, wherein the compound has from 4 to 9 carbon atoms. In someembodiments, the C₄₋₉ carboxylic acid group contains 4 to 9, 4 to 8, 4to 7, 4 to 6, 4 to 5, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 9, 6 to 8, 6to 7, 7 to 9, 7 to 8, or 8 to 9 carbon atoms. Exemplary C₄₋₉ carboxylicacids include butanoic acid (i.e., butanoate), pentanoic acid (i.e.,pentanoate), hexanoic acid (i.e., hexanoate), heptanoic acid (i.e.,heptanoate), octanoic acid (e.g., octanoate), nonanoic acid (i.e.,nonanoate), 2-methylheptanoic acid (i.e., 2-methylheptanoate),3-methylheptanoic acid (i.e., 3-methylheptanoate), 4-methylheptanoicacid (i.e., 4-methylheptanoate), 5-methylheptanoic acid (i.e.,5-methylheptanoate), and 6-methylheptanoic acid (i.e.,6-methylheptanoate). In some embodiments, the C₄₋₉ carboxylic acid isheptanoate (i.e., heptanoic acid).

As used herein, the term “(C₃₋₈ alkyl)-C(═O)OCH₃ ester” refers to acompound having the following formula:

As used herein, the term “C₃₋₈ alkyl” refers to a saturated hydrocarbongroup that may be straight-chain or branched, having 3 to 8 carbonatoms. In some embodiments, the alkyl group contains 4 to 8, 4 to 7, 4to 6, 4 to 5, 5 to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8, carbonatoms. Example alkyl moieties include n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, n-hexyl, n-heptyl, andn-octyl. In some embodiments, the C₃₋₈ alkyl is a group of the followingformula:

In some embodiments,

a) enzymatically converting a C₄₋₉ carboxylic acid to a (C₃₋₈alkyl)-C(═O)OCH₃ ester; and

b) enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

In some embodiments, the C₄₋₉ carboxylic acid is enzymatically convertedto the (C₃₋₈ alkyl)-C(═O)OCH₃ ester using a polypeptide having fattyacid O-methyltransferase activity. In some embodiments, the polypeptidehaving fatty acid O-methyltransferase activity has at least 70% sequenceidentity to an amino acid sequence set forth in SEQ ID NO:23, SEQ IDNO:24, or SEQ ID NO:25.

In some embodiments, the (C₃₋₈ alkyl)-C(═O)OCH₃ ester is enzymaticallyconverted to the (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester using a polypeptidehaving monooxygenase activity. In some embodiments, the monooxygenase isclassified under EC 1.14.14.- or EC 1.14.15.-. In some embodiments, saidthe monooxygenase has at least 70% sequence identity to an amino acidsequence set forth in SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ IDNO:28 and/or SEQ ID NO:29.

In some embodiments, the C₄₋₉ carboxylic acid is enzymatically producedfrom C₄₋₉ alkanoyl-CoA. As used herein, the term “C₄₋₉ alkanoyl-CoA”refers to a compound having the following formula:

wherein the C₃₋₈ alkyl group is as defined herein. In some embodiments,the C₃₋₈ alkyl is a group of the following formula:

In some embodiments, a polypeptide having thioesterase activityenzymatically produces the C₄₋₉ carboxylic acid from C₄₋₉ alkanoyl-CoA.In some embodiments, the thioesterase has at least 70% sequence identityto the amino acid sequence set forth in SEQ ID NO:1, and/or SEQ ID NO:33-34.

In some embodiments, a polypeptide having butanal dehydrogenase activityand a polypeptide having aldehyde dehydrogenase activity enzymaticallyproduce the C₄₋₉ carboxylic acid from the C₄₋₉ alkanoyl-CoA.

In some embodiments, the method of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester is a method of producing7-hydroxyheptanoate methyl ester. In some embodiments, the methodcomprises:

a) enzymatically converting heptanoate to heptanoate methyl ester; and

b) enzymatically converting the heptanoate methyl ester to7-hydroxyheptanoate methyl ester.

In some embodiments, heptanoate is enzymatically converted to heptanoatemethyl ester using a polypeptide having fatty acid O-methyltransferaseactivity. In some embodiments, the polypeptide having fatty acidO-methyltransferase activity has at least 70% sequence identity to anamino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, or SEQ IDNO:25.

In some embodiments, heptanoate methyl ester is enzymatically convertedto 7-hydroxyheptanoate methyl ester using a polypeptide havingmonooxygenase activity. In some embodiments, the polypeptide havingmonooxygenase activity has at least 70% sequence identity to an aminoacid sequence set forth in SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16,SEQ ID NO:28 and/or SEQ ID NO:29. In some embodiments, said themonooxygenase is classified under EC 1.14.14.- or EC 1.14.15.-.

In some embodiments, heptanoate is enzymatically produced fromheptanoyl-CoA. In some embodiments, a polypeptide having thioesteraseactivity enzymatically produces heptanoate from heptanoyl-CoA. In someembodiments, the polypeptide having thioesterase activity has at least70% sequence identity to the amino acid sequence set forth in SEQ IDNO:1, and/or SEQ ID NO: 33-34.

In some embodiments, a butanal dehydrogenase and an aldehydedehydrogenase enzymatically produce heptanoate from heptanoyl-CoA.

The present application further provides methods of producing one ormore terminal hydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl)esters. As used herein, the term “(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl)ester” refers to a compound having the following formula:

wherein the C₃₋₈ alkyl group is as defined herein. As used herein theterm “terminal hydroxy-substituted “(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl)ester” refers to a compound having the following formula:

wherein at least one of the alkyl groups (i.e., at least one of the C₄₋₉alkyl and C₃₋₈ alkyl groups) is subsequently substituted by at least oneterminal hydroxy (—OH) group, and the C₃₋₈ alkyl is as defined herein.As used herein, the term “C₄₋₉ alkyl” refers to a saturated hydrocarbongroup that may be straight-chain or branched, having 4 to 9 carbonatoms. In some embodiments, the alkyl group contains 4 to 9, 4 to 8, 4to 7, 4 to 6, 4 to 5, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 9, 6 to 8, 6to 7, 7 to 9, 7 to 8, or 8 to 9 carbon atoms. Example alkyl moietiesinclude n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl,neo-pentyl, n-hexyl, n-heptyl, n-octyl, and n-nonyl. In someembodiments, one of the alkyl groups is substituted by at least oneterminal hydroxy group. In some embodiments, each of the alkyl groups issubstituted by at least one terminal hydroxy group. In some embodiments,one of the alkyl groups is substituted by one terminal hydroxy group. Insome embodiments, each of the alkyl groups is substituted by oneterminal hydroxy group. In some embodiments, the terminalhydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester is selectedfrom the group consisting of:

In some embodiments, the method includes:

a) enzymatically converting C₄₋₉ alkanoyl-CoA to a (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester; and

b) enzymatically converting the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esterto any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, or (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester.

In some embodiments, C₄₋₉ alkanoyl-CoA is enzymatically converted to the(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester using a polypeptide havingalcohol O-acetyltransferase activity. In some embodiments, the alcoholO-acetyltransferase has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 26.

In some embodiments, the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester isenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having monooxygenase activity. In some embodiments, thepolypeptide having monooxygenase activity is classified under EC1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further includes enzymaticallyconverting (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester to a C₄₋₉ hydroxyalkanoate. Insome embodiments, a polypeptide having esterase activity enzymaticallyconverts (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester to the C₄₋₉ hydroxyalkanoate.

As used herein, the term C₄₋₉ hydroxyalkanoate refers to a compoundhaving the following formula:

wherein the C₃₋₈ hydroxyalkyl is as defined herein. Example C₄₋₉hydroxyalkanoates include, but are not limited to, 7-hydroxyheptanoate(i.e., 7-hydroxyheptanoic acid), 6-hydroxyheptanoate (i.e.,6-hydroxyheptanoic acid), 5-hydroxyheptanoate (i.e., 5-hydroxyheptanoicacid), and the like. It is understood by those skilled in the art thatthe specific form will depend on pH (e.g., neutral or ionized forms,including any salt forms thereof). In some embodiments, the C₃₋₈hydroxyalkyl is a group having the following formula:

In some embodiments, the method of producing one or morehydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters is a methodof producing one or more heptanoic acid heptyl hydroxyl esters. In someembodiments, the method includes:

a) enzymatically converting heptanoyl-CoA to heptanoic acid heptylester; and

b) enzymatically converting heptanoic acid heptyl ester to any of7-hydroxyheptanoic acid heptyl ester, 7-hydroxyheptanoic acid7-hydroxyheptyl ester, or heptanoic acid 7-hydroxyheptyl ester.

In some embodiments, heptanoyl-CoA is enzymatically converted toheptanoic acid heptyl ester using a polypeptide having alcoholO-acetyltransferase activity. In some embodiments, the polypeptidehaving alcohol O-acetyltransferase activity has at least 70% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, heptanoic acid heptyl ester is enzymaticallyconverted to any of 7-hydroxyheptanoic acid heptyl ester,7-hydroxyheptanoic acid 7-hydroxyheptyl ester and/or heptanoic acid7-hydroxyheptyl ester using a polypeptide having monooxygenase activity.In some embodiments, the polypeptide having monooxygenase activity isclassified under EC 1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further includes enzymaticallyconverting 7-hydroxyheptanoic acid 7-hydroxyheptyl ester or7-hydroxyheptanoic acid heptyl ester to 7-hydroxyheptanoate. In someembodiments, a polypeptide having esterase activity enzymaticallyconverts 7-hydroxyheptanoic acid 7-hydroxyheptyl ester or7-hydroxyheptanoic acid heptyl ester to 7-hydroxyheptanoate.

In some embodiments, the method further includes enzymaticallyconverting 7-hydroxyheptanoic acid 7-hydroxyheptyl ester or heptanoicacid 7-hydroxyheptyl ester to 1,7-heptanediol. In some embodiments, apolypeptide having esterase activity enzymatically converts7-hydroxyheptanoic acid 7-hydroxyheptyl ester or heptanoic acid7-hydroxyheptyl ester to 1,7-heptanediol.

In some embodiments, the method can include enzymatically converting7-hydroxyheptanoic acid heptyl ester, 7-hydroxyheptanoic acid7-hydroxyheptyl ester, or heptanoic acid 7-hydroxyheptyl ester to7-hydroxyheptanoate and/or 1,7-heptanediol. In some embodiments, apolypeptide having esterase activity enzymatically converts7-hydroxyheptanoic acid heptyl ester, 7-hydroxyheptanoic acid7-hydroxyheptyl ester, or heptanoic acid 7-hydroxyheptyl ester to7-hydroxyheptanoate and/or 1,7-heptanediol.

In some embodiments, the method further includes enzymaticallyconverting 1,7-heptanediol to 7-hydroxyheptanal. In some embodiments, apolypeptide having alcohol dehydrogenase activity enzymatically converts1,7-heptanediol to 7-hydroxyheptanal.

In some embodiments, the method further includes enzymaticallyconverting 7-hydroxyheptanal to 7-hydroxyheptanoate. In someembodiments, a polypeptide having aldehyde dehydrogenase activityenzymatically converts 7-hydroxyhexanal to 7-hydroxyheptanoate.

In some embodiments, the method further includes enzymaticallyconverting 7-hydroxyheptanoate methyl ester to 7-hydroxyheptanoate. Insome embodiments, a polypeptide having demethylase activity or apolypeptide having esterase activity enzymatically converts7-hydroxyheptanoate methyl ester to 7-hydroxyheptanoate.

In some embodiments, the method further includes enzymaticallyconverting 7-hydroxyheptanoate to a product selected from the groupconsisting of pimelic acid, pimelate semialdehyde, 7-aminoheptanoate,heptamethylenediamine, and 1,7-heptanediol.

In some embodiments, the method includes enzymatically converting7-hydroxyheptanoate to pimelate semialdehyde using a polypeptide havingalcohol dehydrogenase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, or a polypeptide having monooxygenase activity.

In some embodiments, the method further includes enzymaticallyconverting pimelate semialdehyde to pimelic acid using a polypeptidehaving 5-oxopentanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having monooxygenase activity.

In some embodiments, the method further includes enzymaticallyconverting pimelate semialdehyde to 7-aminoheptanoate. In someembodiments, a ω-transaminase enzymatically converts pimelatesemialdehyde to 7-aminoheptanoate.

In some embodiments, the method further includes enzymaticallyconverting 7-aminoheptanoate to heptamethylenediamine. In someembodiments, the method further includes enzymatically convertingpimelate semialdehyde to heptamethylenediamine. In some embodiments,pimelate semialdehyde or 7-aminoheptanoate is enzymatically converted toheptamethylenediamine using a polypeptide having carboxylate reductaseactivity and/or a polypeptide having ω-transaminase activity andoptionally one or more of a polypeptide having N-acetyl transferaseactivity, a polypeptide having acetylputrescine deacetylase activity,and a polypeptide having alcohol dehydrogenase activity.

In some embodiments, 7-hydroxyheptanoate is enzymatically converted to1,7-heptanediol using a polypeptide having carboxylate reductaseactivity and a polypeptide having alcohol dehydrogenase activity.

In some embodiments, the method further comprises enzymaticallyconverting 1,7-heptanediol to heptamethylenediamine. In someembodiments, a polypeptide having alcohol dehydrogenase activity and apolypeptide having ω-transaminase activity enzymatically converts1,7-heptanediol to heptamethylenediamine.

In some embodiments, a polypeptide having carboxylate reductaseactivity, a polypeptide having ω-transaminase activity, and apolypeptide having alcohol dehydrogenase activity enzymatically converts7-hydroxyheptanoate to heptamethylenediamine.

In some embodiments, the ω-transaminase has at least 70% sequenceidentity to any one of the amino acid sequences set forth in SEQ ID NO.8-13.

In some embodiments, heptanoyl-CoA is produced from acetyl-CoA andpropanoyl-CoA via two cycles of CoA-dependent carbon chain elongation.In some embodiments, each of said two cycles of CoA-dependent carbonchain elongation comprises using a polypeptide having β-ketothiolaseactivity or a polypeptide having acetyl-CoA carboxylase activity and apolypeptide having β-ketoacyl-[acp] synthase activity, a polypeptidehaving 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to form heptanoyl-CoA from acetyl-CoA and propanoyl-CoA.

Enzymes Converting Heptanoate or Heptanoyl-CoA to 7-Hydroxyheptanoate

As depicted in FIG. 7, heptanoate methyl ester can be formed fromheptanoate using a polypeptide having fatty acid O-methyltransferaseactivity, such as the fatty acid O-methyltransferase classified, forexample, under EC 2.1.1.15. For example, the polypeptide having fattyacid O-methyltransferase activity can be obtained from Mycobacteriummarinum (GenBank Accession No. ACC41782.1. SEQ ID NO:23), Mycobacteriumsmegmatis (see GenBank Accession No. ABK73223.1, SEQ ID NO: 24), orPseudomonas putida (see GenBank Accession No. CAA39234.1, SEQ ID NO:25).

Heptanoate methyl ester can be converted to 7-hydroxyheptanoate methylester using a polypeptide having monooxygenase activity classified, forexample, under EC 1.14.14.- or EC 1.14.15.- (e.g., EC 1.14.15.1 or EC1.14.15.3) For example, a polypeptide having monooxygenase activity canbe, for example, from the CYP153A family (SEQ ID NOs:14-16), the CYP52A3family (See Genbank Accession No: BAA00371.1, SEQ ID NO: 29) or the alkBfamily such as the gene product of alkBGT from Pseudomonas putida (SeeGenbank Accession No. CAB51047.1, SEQ ID NO: 28). See, FIG. 7.

7-hydroxyheptanoate methyl ester can be converted to 7-hydroxyheptanoateusing a polypeptide having demethylase activity classified, for example,under EC 2.1.1.- such as the gene product of ligM (see GenBank AccessionNo. BAD61059.1; SEQ ID NO:31) or desA (GenBank Accession No. BAC79257.1;SEQ ID NO:32) or using a polypeptide having esterase activityclassified, for example under EC 3.1.1.- such as the gene product ofEstC (see GenBank Accession No. AAC60471.2. SEQ ID NO: 27) (see GenBankAccession No. AAC60471.2, SEQ ID NO: 27).

As depicted in FIG. 7, heptanoyl-CoA can be converted to heptanoic acidheptyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.84)such as the gene product of Eht1 (Genbank Accession No: CAA85138.1, SEQID NO: 26).

Heptanoic acid heptyl ester can be converted to 7-hydroxyheptanoic acidheptyl ester and/or 7-hydroxyheptanoic acid 7-hydroxyheptyl ester usinga polypeptide having monooxygenase activity classified, for example,under EC 1.14.14.- or EC 1.14.15.- (e.g., EC 1.14.15.1 or EC 1.14.15.3)For example, a polypeptide having monooxygenase activity can be, forexample, from the CYP153A family, the CYP52A3 family (Genbank AccessionNo: BAA00371.1, SEQ ID NO: 29) or the alkB family such as the geneproduct of alkBGT from Pseudomonas putida (Genbank Accession No.CAB51047.1, SEQ ID NO: 28). See, FIG. 7.

7-hydroxyheptanoic acid heptyl ester and 7-hydroxyheptanoic acid7-hydroxyheptyl can be converted to 7-hydroxyheptanoate using apolypeptide having esterase activity classified, for example, under EC3.1.1.- (EC 3.1.1.1 or EC 3.1.1.6) such as the gene product of EstC (seeGenBank Accession No. AAC60471.2, SEQ ID NO: 27).

For example, the monooxygenase CYP153A family classified, for example,under EC 1.14.15.- (e.g., EC 1.14.15.1 or EC 1.14.15.3) is soluble andhas regio-specificity for terminal hydroxylation, accepting medium chainlength substrates (see, e.g., Koch et al., Appl. Environ. Microbiol.,2009, 75(2), 337-344; Funhoff et al., 2006, J. Bacteriol., 188(44):5220-5227; Van Beilen & Funhoff, Current Opinion in Biotechnology, 2005,16, 308-314; Nieder and Shapiro, J. Bacteriol., 1975, 122(1), 93-98).Although non-terminal hydroxylation is observed in vitro for CYP153A, invivo only 1-hydroxylation occurs (see, Funhoff et al., 2006, supra).

The substrate specificity and activity of terminal monooxygenases hasbeen broadened via successfully, reducing the chain length specificityof CYP153A to below C8 (Koch et al., 2009, supra).

In some embodiments, heptanoate can be enzymatically formed fromheptanoyl-CoA using a polypeptide having thioesterase activityclassified under EC 3.1.2.-, such as the gene product of YciA, tesB(GenBank Accession No. AAA24665.1, SEQ ID NO: 1) or Acot13 (Cantu etal., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry,2008, 47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991, 266(17):11044-11050), the acyl-[acp] thioesterase from a Lactobacillus brevis(GenBank Accession No. ABJ63754.1, SEQ ID NO:33), or a Lactobacillusplantarum (GenBank Accession No. CCC78182.1, SEQ ID NO:34). Suchacyl-[acp] thioesterases have C6-C8 chain length specificity (see, forexample, Jing et al., 2011, BMC Biochemistry, 12(44)). See, FIG. 3.

In some embodiments, heptanoate can be enzymatically formed fromheptanoyl-CoA using a polypeptide having butanal dehydrogenase activityclassified, for example, under EC 1.2.1.57 (see, e.g. GenBank AccessionNo. AAD39015, SEQ ID NO:30) or an aldehyde dehydrogenase classified, forexample, under EC 1.2.1.4 (see, Ho & Weiner, J. Bacteriol., 2005,187(3):1067-1073). See, FIG. 3.

Enzymes Generating Heptanoyl-CoA for Conversion to a C7 Building Block

As depicted in FIG. 1 and FIG. 2, heptanoyl-CoA can be formed fromacetyl-CoA or propanoyl-CoA via two cycles of CoA-dependent carbon chainelongation using either NADH or NADPH dependent enzymes.

In some embodiments, a CoA-dependent carbon chain elongation cyclecomprises using a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity and a polypeptide having trans-2-enoyl-CoA reductaseactivity. A polypeptide having β-ketothiolase activity can convertpropanoyl-CoA to 3-oxopentanoyl-CoA and can convert pentanoyl-CoA to3-oxoheptanoyl-CoA. A polypeptide having acetyl-CoA carboxylase activitycan convert acetyl-CoA to malonyl-CoA. A polypeptide havingacetoacetyl-CoA synthase activity can convert malonyl-CoA toacetoacetyl-CoA. A polypeptide having 3-hydroxybutyryl-CoA dehydrogenaseactivity can convert 3-oxopentanoyl-CoA to 3-hydroxypentanoyl CoA. Apolypeptide having 3-oxoacyl-CoA reductase/3-hydroxyacyl-CoAdehydrogenase activity can convert 3-oxoheptanoyl-CoA to3-hydroxyheptanoyl-CoA. A polypeptide having enoyl-CoA hydrataseactivity can convert 3-hydroxypentanoyl-CoA to pent-2-enoyl-CoA and canconvert 3-hydroxyheptanoyl-CoA to hept-2-enoyl-CoA. A polypeptide havingtrans-2-enoyl-CoA reductase activity can convert hept-2-enoyl-CoA toheptanoyl-CoA and can convert hept-2-enoyl-CoA to heptanoyl-CoA. See.FIGS. 1 and 2.

In some embodiments, a polypeptide having β-ketothiolase activity can beclassified under EC 2.3.1.16, such as the gene product of bktB (See,e.g., Genbank Accession AAC38322.1). The polypeptide havingβ-ketothiolase activity encoded by bktB from Cupriavidus necator acceptspropanoyl-CoA and pentanoyl-CoA as substrates. When pentanoyl-CoA is thesubstrate, the CoA-activated C7 aliphatic backbone (3-oxoheptanoyl-CoA)is produced (see, e.g., Haywood et al., FEMS Microbiology Letters, 1988,52:91-96; Slater et al., J. Bacteriol., 1998, 180(8):1979-1987). Thepolypeptide having β-ketothiolase activity encoded by paaJ (See, e.g.,Genbank Accession No. AAC74479.1), catF and pcaF can be classifiedunder, for example, EC 2.3.1.174. The polypeptide having β-ketothiolaseactivity encoded by paaJ condenses acetyl-CoA and succinyl-CoA to3-oxoadipyl-CoA (see, for example, Fuchs et al., 2011, Nature ReviewsMicrobiology, 9, 803-816; Gobel et al., 2002, J. Bacteriol., 184(1),216-223) See FIGS. 1 and 2.

In some embodiments, a polypeptide having acetyl-CoA carboxylaseactivity can be classified, for example, under EC 6.4.1.2. In someembodiments, a polypeptide having β-ketoacyl-[acp] synthase activity canbe classified, for example, under 2.3.1.180 such as the gene product ofFabH from Staphylococcus aereus (Qiu et al., 2005, Protein Science, 14:2087-2094). See, FIGS. 1 and 2.

In some embodiments, a polypeptide having 3-hydroxyacyl-CoAdehydrogenase activity or a polypeptide having 3-oxoacyl-CoAdehydrogenase activity can be classified under EC 1.1.1.-. For example,the polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity can beclassified under EC 1.1.1.35, such as the gene product of fadB (FIG. 1);classified under EC 1.1.1.157, such as the gene product of hbd (can bereferred to as a 3-hydroxybuyryl-CoA dehydrogenase) (FIG. 1); orclassified under EC 1.1.1.36, such as the acetoacetyl-CoA reductase geneproduct of phaB (Liu & Chen, Appl. Microbiol. Biotechnol., 2007,76(5):1153-1159; Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915; Budde et al., J. Bacteriol., 2010, 192(20):5319-5328)(FIG. 2).

In some embodiments, a polypeptide having 3-oxoacyl-CoA reductaseactivity can be classified under EC 1.1.1.100, such as the gene productof fabG (Budde et al., J. Bacteriol., 2010, 192(20):5319-5328; Nomura etal., Appl. Environ. Microbiol., 2005, 71(8):4297-4306). See, FIG. 2.

In some embodiments, a polypeptide having enoyl-CoA hydratase activitycan be classified under EC 4.2.1.17, such as the gene product of crt(Genbank Accession No. AAA95967.1) (FIG. 1), or classified under EC4.2.1.119, such as the gene product of phaJ (Genbank Accession No.BAA21816.1) (FIG. 2) (Shen et al., 2011, supra; Fukui et al., J.Bacteriol., 1998, 180(3):667-673).

In some embodiments, a polypeptide having trans-2-enoyl-CoA reductaseactivity can be classified under EC 1.3.1.38 (FIG. 2), EC 1.3.1.8 (FIG.2), or EC 1.3.1.44 (FIG. 1), such as the gene product of ter (GenbankAccession No. AAW66853.1) (Nishimaki et al., J. Biochem., 1984,95:1315-1321; Shen et al., 2011, supra) or tdter (Genbank Accession No.AAS11092.1) (Bond-Watts et al., Biochemistry, 2012, 51:6827-6837).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of aC7 Building Block

As depicted in FIGS. 4, 5, and 7, a terminal carboxyl group can beenzymatically formed using a polypeptide having thioesterase activity, apolypeptide having aldehyde dehydrogenate activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide havingmonooxygenase activity, a polypeptide having esterase activity, or apolypeptide having demethylase activity.

In some embodiments, the first terminal carboxyl group is enzymaticallyformed by a polypeptide having syringate O-demethylase activityclassified under EC 2.1.1.- such as the gene products of ligM (seeGenBank Accession No. BAD61059.1; SEQ ID NO:31) or desA (GenBankAccession No. BAC79257.1; SEQ ID NO:32) or a polypeptide having esteraseactivity classified under EC 3.1.1.- such as the gene product of EstC(see, e.g., GenBank Accession No. AAC60471.2, SEQ ID NO: 27) See, e.g.,FIG. 7.

In some embodiments, the first terminal carboxyl group is enzymaticallyformed by a polypeptide having aldehyde dehydrogenase activityclassified, for example, under EC 1.2.1.3 or EC 1.2.1.4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a polypeptidehaving aldehyde dehydrogenase activity classified, for example, under EC1.2.1.3 (see, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81,185-192). See FIG. 4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a polypeptidehaving dehydrogenase activity classified under EC 1.2.1.- such as apolypeptide having 5-oxopentanoate dehydrogenase activity (e.g., thegene product of CpnE), a polypeptide having 6-oxohexanoate dehydrogenaseactivity (e.g., the gene product of ChnE from Acinetobacter sp.), apolypeptide having 7-oxoheptanoate dehydrogenase activity (e.g., thegene product of ThnG from Sphingomonas macrogolitabida) (Iwaki et al.,App. Environ. Microbiol., 1999, 65(11), 5158-5162; López-Sánchez et al.,Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, apolypeptide having 5-oxopentanoate dehydrogenase activity can beclassified under EC 1.2.1.20. For example, a polypeptide having6-oxohexanoate dehydrogenase activity can be classified under EC1.2.1.63. For example, a polypeptide having 7-oxoheplanoatedehydrogenase activity can be classified under EC 1.2.1.-. See, e.g.,FIG. 4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a polypeptidehaving monooxygenase activity in the cytochrome P450 family such asCYP4F3B (see, e.g., Sanders et al., J. Lipid Research, 2005, 46(5):1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6):2064-2071).See, e.g., FIG. 4.

The utility of ω-oxidation in introducing carboxyl groups into alkaneshas been demonstrated in the yeast Candida tropicalis, leading to thesynthesis of adipic acid (Okuhara et al., Agr. Biol. Chem., 1971, 35(9),1376-1380).

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of a C7Building Block

As depicted in FIG. 5 and FIG. 6, terminal amine groups can beenzymatically formed using a polypeptide having ω-transaminase activityor a polypeptide having deacetylase activity.

In some embodiments, the first terminal amine group leading to thesynthesis of 7-aminoheptanoic acid, 7-aminoheptanal, or 7-aminoheptanolis enzymatically formed by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.- such as EC 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as that obtainedfrom Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ IDNO: 8), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ IDNO: 9), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ IDNO: 10), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQID NO: 11), Vibrio fluvialis (Genbank Accession No. AAA57874.1, SEQ IDNO: 13), Streptomyces griseus, or Clostridium viride. An additionalpolypeptide having ω-transaminase activity that can be used in themethods and hosts described herein is from Escherichia coli (GenbankAccession No. AAA57874.1, SEQ ID NO: 12). Some of the polypeptideshaving ω-transaminase activity classified, for example, under EC2.6.1.29 or EC 2.6.1.82 are polypeptides having diamine ω-transaminaseactivity (e.g., SEQ ID NO: 12). See, e.g., FIGS. 5 and 6.

The reversible polypeptide having ω-transaminase activity fromChromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO:8) has demonstrated analogous activity accepting 6-aminohexanoic acid asamino donor, thus forming the first terminal amine group in adipatesemialdehyde (Kaulmann et al., Enzyme and Microbiol Technology, 2007,41, 628-637).

The reversible polypeptide having 4-aminobutyrate:2-oxoglutaratetransaminase activity from Streptomyces griseus has demonstratedanalogous activity for the conversion of 6-aminohexanoate to adipatesemialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146, 101-106).

The reversible polypeptide having 5-aminovalerate transaminase activityfrom Clostridium viride has demonstrated analogous activity for theconversion of 6-aminohexanoate to adipate semialdehyde (Barker et al.,J. Biol. Chem., 1987, 262(19), 8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by apolypeptide having diamine transaminase activity. For example, thesecond terminal amino group can be enzymatically formed by a polypeptidehaving diamine transaminase activity classified, for example, under EC2.6.1.-, e.g., EC 2.6.1.29 or classified, for example, under EC2.6.1.82, such as the gene product of YgjG from E. coli (GenbankAccession No. AAA57874.1, SEQ ID NO: 12). See, e.g., FIG. 6.

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The polypeptide having diamine transaminase activity from E. coli strainB has demonstrated activity for 1,7 diaminoheptane (Kim, The Journal ofChemistry, 1964, 239(3), 783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by apolypeptide having deacetylase activity classified, for example, underEC 3.5.1.62 such as a polypeptide having acetylputrescine deacetylaseactivity. The polypeptide having acetylputrescine deacetylase activityfrom Micrococcus luteus K-11 accepts a broad range of carbon chainlength substrates, such as acetylputrescine, acetylcadaverine andN⁸-acetylspermidine (see, for example, Suzuki et al., 1986, BBA—GeneralSubjects, 882(1): 140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of aC7 Building Block

As depicted in FIG. 8, a terminal hydroxyl group can be enzymaticallyforming using a polypeptide having alcohol dehydrogenase activity. Forexample, the second terminal hydroxyl group leading to the synthesis of1,7 heptanediol is enzymatically formed by a polypeptide having alcoholdehydrogenase activity classified under EC 1.1.1.- (e.g., EC 1.1.1.1,1.1.1.2, 1.1.1.21, or 1.1.1.184).

A first terminal hydroxyl group can be enzymatically formed with apolypeptide having monoxygenase activity as discussed above with respectto the formation of 7-hydroxyheptanoate methyl ester in FIG. 7.

As depicted in FIG. 8, heptanoyl-CoA can be converted to heptanoic acidheptyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.- (84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 26).

Heptanoic acid heptyl ester can be converted to 7-hydroxyheptanoic acid7-hydroxyheptyl ester using a polypeptide having monooxygenase activityclassified, for example, under EC 1.14.14.- or EC 1.14.15.- (EC1.14.15.1 or EC 1.14.15.3). Heptanoic acid heptyl ester can be convertedto heptanoic acid 7-hydroxyheptyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (1,3). For example, a polypeptide having monooxygenaseactivity can be, for example, from the CYP153A family, the CYP52A3family or the alkB family such as the gene product of alkBGT fromPseudomonas putida. See, e.g., FIG. 7.

Heptanoic acid 7-hydroxyheptyl ester and 7-hydroxyheptanoic acid7-hydroxyheptyl can be converted to 1,7-heptanediol using a polypeptidehaving esterase activity classified, for example, under EC 3.1.1.-(e.g., EC 3.1.1.1 or EC 3.1.1.6) such as the gene product of EstC (seeGenBank Accession No. AAC60471.2, SEQ ID NO: 27).

Biochemical Pathways Pathways to Propanoyl-CoA

In some embodiments, propanoyl-Coenzyme A (CoA) is a precursor leadingto one or more central precursors in the synthesis of one or more C7building blocks (see, e.g., FIG. 9).

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite succinyl-CoA by conversion of succinyl-CoA to(2R)-methylmalonyl-CoA by a polypeptide having methylmalonyl-CoA mutaseactivity classified, for example, under EC 5.4.99.2; followed byconversion to (2S)-methylmalonyl-CoA by a polypeptide havingmethylmalonyl-CoA epimerase activity classified, for example, under EC5.1.99.1; followed by conversion to propanoyl-CoA by a polypeptidehaving methylmalonyl-CoA carboxytransferase activity classified, forexample, under EC 2.1.3.1 or a polypeptide having methylmalonyl-CoAdecarboxylase activity classified, for example, under EC 4.1.1.41. Seee.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite, L-threonine, by conversion of L-threonine to 2-oxobutyrateby a polypeptide having threonine ammonia lyase activity classified, forexample, under EC 4.3.1.19; followed by conversion to propanoyl-CoA by apolypeptide having 2-ketobutyrate formate-lyase activity classified, forexample, under EC 2.3.1.- such as the gene product of tdcE (Tseng etal., Microbiol Cell Factories, 2010, 9:96). See, e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from 1,2-propanediolby conversion to propanal by a polypeptide having propanedioldehydratase activity classified, for example, under EC 4.2.1.28;followed by conversion to propanoyl-CoA by a polypeptide havingCoA-dependent propionaldehyde dehydrogenase activity such as the geneproduct of pduP (Luo et al., Bioresource Technology, 2012, 103:1-6).See, e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the carbonsource, levulinic acid, by conversion of levulinic acid to levulinyl-CoAby a polypeptide having acyl-CoA synthetase or ligase activityclassified, for example, under EC 6.2.1.-, followed by conversion topropanoyl-CoA by a polypeptide having transferase activity classified,for example, under EC 2.3.1.- (Jaremko and Yu, J. Biotechnol., 2011,155:293-298). See, e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite, pyruvate, by conversion of pyruvate to L-lactate by apolypeptide having L-lactate dehydrogenase activity classified, forexample, under EC 1.1.1.27; followed by conversion to lactoyl-CoA by apolypeptide having proprionate CoA-transferase activity classified, forexample, under EC 2.8.3.1; followed by conversion to propenoyl-CoA by apolypeptide having lactoyl-CoA dehydratase activity classified, forexample, under EC 4.2.1.54; followed by conversion to propanoyl-CoA by apolypeptide having butyryl-CoA dehydrogenase activity classified, forexample, under EC 1.3.8.1 or a polypeptide having medium-chain acyl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.7. See,e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite, malonyl-CoA, by conversion of malonyl-CoA to malonatesemialdehyde by a polypeptide having malonyl-CoA reductase activityclassified, for example, under EC 1.2.1.75; followed by conversion to3-hydroxypropionate by a polypeptide having 3-hydroxypropionatedehydrogenase activity classified, for example, under EC 1.1.1.59;followed by conversion to 3-hydroxypropionyl-CoA by a polypeptide having3-hydroxyisobutyryl-CoA hydrolase activity classified, for example,under EC 6.2.1.- such as EC 6.2.1.36; followed by conversion topropenoyl-CoA by a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity classified, for example, under EC 4.2.1.116; followed byconversion to propanoyl-CoA by a polypeptide having butyryl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.1 or apolypeptide having medium-chain acyl-CoA dehydrogenase activityclassified, for example, under EC 1.3.8.7. See, e.g., FIG. 9.

Pathways to Heptanoyl-CoA as Central Precursor to C7 Building Blocks

In some embodiments, heptanoyl-CoA is synthesized from propanoyl-CoA byconversion of propanoyl-CoA to 3-oxopentanoyl-CoA by a polypeptidehaving β-ketothiolase activity classified, for example, under EC2.3.1.16, such as the gene product of bktB (Genbank Accession No.AAC38322.1) or classified, for example, under EC 2.3.1.174 such as thegene product of paaJ (Genbank Accession No. AAC74479.1); followed byconversion of 3-oxopentanoyl-CoA to (S) 3-hydroxybutanoyl-CoA by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.35, such as the gene product of fadB orclassified, for example, under EC 1.1.1.157 such as the gene product ofhbd; followed by conversion of (S) 3-hydroxypentanoyl-CoA topent-2-enoyl-CoA by a polypeptide having enoyl-CoA hydratase activityclassified, for example, under EC 4.2.1.17 such as the gene product ofcrt (Genbank Accession No. AAA95967.1); followed by conversion ofpent-2-enoyl-CoA to pentanoyl-CoA by a polypeptide havingtrans-2-enoyl-CoA reductase activity classified, for example, under EC1.3.1.44 such as the gene product of ter (Genbank Accession No.AAW66853.1) or tdter (Genbank Accession No. AAS11092.1); followed byconversion of pentanoyl-CoA to 3-oxo-heptanoyl-CoA by a polypeptidehaving β-ketothiolase activity classified, for example, under EC2.3.1.16 such as the gene product of bktB (Genbank Accession No.AAC38322.1) or classified, for example, under EC 2.3.1.174 such as thegene product of paaJ (Genbank Accession No. AAC74479.1); followed byconversion of 3-oxo-heptanoyl-CoA to (S) 3-hydroxyheptanoyl-CoA by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.35 such as the gene product of fadB or by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.157 such as the gene product of hbd;followed by conversion of (S) 3-hydroxyheptanoyl-CoA to hept-2-enoyl-CoAby a polypeptide having enoyl-CoA hydratase activity classified, forexample, under EC 4.2.1.17 such as the gene product of crt (GenbankAccession No. AAA95967.1); followed by conversion of hept-2-enoyl-CoA toheptanoyl-CoA by a polypeptide having trans-2-enoyl-CoA reductaseactivity classified, for example, under EC 1.3.1.44 such as the geneproduct of ter (Genbank Accession No. AAW66853.1) or tdter (GenbankAccession No. AAS11092.1). See FIG. 1.

In some embodiments, heptanoyl-CoA is synthesized from the centralmetabolite, propanoyl-CoA, by conversion of propanoyl-CoA to3-oxopentanoyl-CoA by a polypeptide having β-ketothiolase activityclassified, for example, under EC 2.3.1.16, such as the gene product ofbktB; followed by conversion of 3-oxopentanoyl-CoA to (R)3-hydroxypentanoyl-CoA by a polypeptide having 3-oxoacyl-CoA reductaseactivity classified, for example, under EC 1.1.1.100, such as the geneproduct of fadG or by a polypeptide having acetoacetyl-CoA reductaseactivity classified, for example, under EC 1.1.1.36 such as the geneproduct of phaB; followed by conversion of (R) 3-hydroxypentanoyl-CoA topent-2-enoyl-CoA by a polypeptide having enoyl-CoA hydratase activityclassified, for example, under EC 4.2.1.119 such as the gene product ofphaJ (Genbank Accession No. BAA21816.1); followed by conversion ofpent-2-enoyl-CoA to pentanoyl-CoA by a polypeptide havingtrans-2-enoyl-CoA reductase activity classified, for example, under EC1.3.1.38 or a polypeptide having acyl-CoA dehydrogenase activityclassified, for example, under EC 1.3.1.8; followed by conversion ofpentanoyl-CoA to 3-oxo-heptanoyl-CoA by a polypeptide havingβ-ketothiolase activity classified, for example, under EC 2.3.1.16 suchas the gene product of bktB (Genbank Accession No. AAC38322.1) orclassified, for example, under EC 2.3.1.174 such as the gene product ofpaaJ (Genbank Accession No. AAC74479.1); followed by conversion of3-oxo-heptanoyl-CoA to (R) 3-hydroxyheptanoyl-CoA by a polypeptidehaving 3-oxoacyl-CoA reductase activity classified, for example, underEC 1.1.1.100 such as the gene product of fabG; followed by conversion of(R) 3-hydroxyheptanoyl-CoA to hept-2-enoyl-CoA by a polypeptide havingenoyl-CoA hydratase activity classified, for example, under EC 4.2.1.119such as the gene product of phaJ (Genbank Accession No. BAA21816.1);followed by conversion of hept-2-enoyl-CoA to heptanoyl-CoA by apolypeptide having trans-2-enoyl-CoA reductase activity classified, forexample, under EC 1.3.1.38 or a polypeptide having acyl-CoAdehydrogenase activity classified, for example, under EC 1.3.1.8. SeeFIG. 2.

In some embodiments, 3-oxopentanoyl-CoA can be synthesized fromacetyl-CoA. A polypeptide having acetyl-CoA carboxylase activityclassified, for example, under EC 6.4.1.2 can be used to convertacetyl-CoA to malonyl-CoA, which can be converted to 3-oxopentanoyl-CoAusing a polypeptide having β-ketoacyl-[acp] synthase activityclassified, for example, under EC 2.3.1.- such as EC 2.3.1.41, EC2.3.1.179 or EC 2.3.1.180 such as the gene product of fabH. See, FIG. 1and FIG. 2.

Pathways Using Heptanoyl-CoA to Produce the Central Precursor Heptanoate

In some embodiments, heptanoate is synthesized from heptanoyl-CoA byconversion of heptanoyl-CoA to heptanoate by a polypeptide havingthioesterase activity classified, for example, under EC 3.1.2.- such asthe gene product of YciA, tesB, Acot13, a Lactobacillus brevisacyl-[acp] thioesterase (GenBank Accession No. ABJ63754.1, SEQ ID NO:33)or a Lactobacillus plantarum acyl-[acp] thioesterase (GenBank AccessionNo. CCC78182.1, SEQ ID NO:34). See, FIG. 3.

In some embodiments, heptanoyl-CoA is converted to heptanal by apolypeptide having butanal dehydrogenase activity classified, forexample, under EC 1.2.1.57 (see, e.g, GenBank Accession No. AAD39015,SEQ ID NO:30); followed by conversion of heptanal to heptanoate by apolypeptide having aldehyde dehydrogenase activity classified, forexample, under EC 1.2.1.4 or EC 1.2.1.3. See FIG. 3.

The conversion of hexanoyl-CoA to hexanal has been demonstrated usingboth NADH and NADPH as co-factors (see Palosaari and Rogers, J.Bacteriol., 1988, 170(7): 2971-2976).

Pathways Using Heptanoyl-CoA to Produce the Central Precursor Heptanol

In some embodiments, heptanoate is synthesized from heptanoyl-CoA byconversion of heptanoyl-CoA to heptanoate by a polypeptide havingthioesterase activity classified, for example, under EC 3.1.2.- such asthe gene product of YciA, tesB or Acot13, a Lactobacillus brevisacyl-[acp] thioesterase (GenBank Accession No. ABJ63754.1, SEQ ID NO:33)or a Lactobacillus plantarum acyl-[acp] thioesterase (GenBank AccessionNo. CCC78182.1, SEQ ID NO:34); followed by conversion of heptanoate toheptanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6, such as the gene product ofcar enhanced by the gene product of sfp; followed by conversion ofheptanal to heptanol by a polypeptide having alcohol dehydrogenaseactivity classified, for example, under EC 1.1.1.- such as EC 1.1.1.1,EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product ofYMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E. coli,GenBank Accession No. AAA69178.1) (see, e.g., Liu et al., Microbiology,2009, 155, 2078-2085; Larroy et al., 2002, Biochem. J., 361(Pt 1),163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257)or the protein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus). See, FIG. 3.

In some embodiments, heptanoyl-CoA is converted to heptanal by apolypeptide having butanal dehydrogenase activity classified, forexample, under EC 1.2.1.57 (see, e.g., GenBank Accession No. BAD61059.1,SEQ ID NO:31); followed by conversion of heptanal to heptanol by apolypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21,or EC 1.1.1.184) such as the gene product of YMR318C (Genbank AccessionNo. CAA90836.1) or YqhD (from E. coli. GenBank Accession No. AAA69178.1)(see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy etal., 2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl.Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBankAccession No. CAA81612.1 (from Geobacillus stearothermophilus). See,FIG. 3.

Pathways Using Heptanoate or Heptanoyl-CoA as Central Precursor to7-Hydroxyheptanoate

In some embodiments, 7-hydroxyheptanoate is synthesized from the centralprecursor, heptanoate, by conversion of heptanoate to heptanoate methylester using a polypeptide having fatty acid O-methyltransferase activityclassified, for example, under EC 2.1.1.15 (e.g., the fatty acidO-methyltransferase from Mycobacterium marinum (GenBank Accession No.ACC41782.1. SEQ ID NO:23), Mycobacterium smegmatis (see GenBankAccession No. ABK73223.1, SEQ ID NO: 24), or Pseudomonas putida (seeGenBank Accession No. CAA39234.1, SEQ ID NO: 25); followed by conversionto 7-hydroxyheptanoate methyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (e.g., EC 1.14.15.1 or EC 1.14.15.3) such as a polypeptidehaving monooxygenase activity in the CYP153A, a CYP52A3 family, or alkBfamily; followed by conversion to 7-hydroxyheptanoate using apolypeptide having syringate O-demethylase activity classified under EC2.1.1.- such as the gene products of ligM (see GenBank Accession No.BAD61059.1; SEQ ID NO:31) or desA (GenBank Accession No. BAC79257.1; SEQID NO:32), or using a polypeptide having esterase activity classifiedunder EC 3.1.1. such as the gene product of EstC (see GenBank AccessionNo. AAC60471.2, SEQ ID NO: 27) (Kim et al., 1994, Biosci. Biotech.Biochem, 58(1), 111-116).

In some embodiments, heptanoate can be enzymatically converted to7-hydroxyheptanoate by a polypeptide having monooxygenase activity(classified, for example, under EC 1.14.14.- or EC 1.14.15.- such as apolypeptide having monooxygenase activity in the CYP153A, the CYP52A3family, and/or the gene product of alkB family.

In some embodiments, heptanoyl-CoA can be converted to heptanoic acidheptyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.- (84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 25);followed by conversion to 7-hydroxyheptanoic acid heptyl ester and/or7-hydroxyheptanoic acid 7-hydroxyheptyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (1,3). For example, a polypeptide having monooxygenaseactivity can be, for example, from the CYP153A family, the CYP52A3family (Genbank Accession No: BAA00371.1, SEQ ID NO: 29) or the alkBfamily such as the gene product of alkBGT from Pseudomonas putida(Genbank Accession No. CAB51047.1, SEQ ID NO: 28); followed byconversion of 7-hydroxyheptanoic acid heptyl ester and/or7-hydroxyheptanoic acid 7-hydroxyheptyl to 7-hydroxyheptanoate using apolypeptide having esterase activity classified, for example, under EC3.1.1.- (1,6) such as the gene product of EstC (see GenBank AccessionNo. AAC60471.2, SEQ ID NO: 27) (Kim et al., 1994, Biosci. Biotech.Biochem, 58(1), 111-116). See FIG. 7.

Pathways Using 7-Hydroxyheptanoate as Central Precursor to Pimelate

Pimelate semialdehyde can be synthesized by enzymatically converting7-hydroxyheptanoate to pimelate semialdehyde using a polypeptide havingalcohol dehydrogenase activity classified, for example, under EC 1.1.1.-such as the gene product of YMR318C (classified, for example, under EC1.1.1.2, see Genbank Accession No. CAA90836.1) (Larroy et al., 2002,Biochem J., 361(Pt 1), 163-172), cpnD (Iwaki et al., 2002, Appl.Environ. Microbiol., 68(11):5671-5684) or gabD (Litke-Eversloh &Steinbuchel, 1999, FEMS Microbiology Letters, 181(1):63-71), apolypeptide having 6-hydroxyhexanoate dehydrogenase activity classified,for example, under EC 1.1.1.258 such as the gene product of ChnD (Iwakiet al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162), or apolypeptide having cytochrome P450 activity (Sanders et al., J. LipidResearch, 2005, 46(5), 1001-1008; Sanders et al., The FASEB Journal,2008, 22(6), 2064-2071). See, FIG. 4. The polypeptide having alcoholdehydrogenase activity encoded by YMR318C has broad substratespecificity, including the oxidation of C7 alcohols.

Pimelate semialdehyde can be enzymatically converted to pimelic acidusing a polypeptide having aldehyde dehydrogenase activity classified,for example, under EC 1.2.1.- (3,16,20,63,79) such as a polypeptidehaving 7-oxoheptanoate dehydrogenase activity (e.g., the gene product ofThnG), a polypeptide having 6-oxohexanoate dehydrogenase activity (e.g.,the gene product of ChnE), or a polypeptide having aldehydedehydrogenase activity classified under EC 1.2.1.3. See FIG. 4.

Pathway Using 7-Hydroxyheptanoate as Central Precursor to7-Aminoheptanoate

In some embodiments, 7-aminoheptanoate is synthesized from7-hydroxyheptanoate by conversion of 7-hydroxyheptanoate to pimelatesemialdehyde using a polypeptide having alcohol dehydrogenase activityclassified, for example, under EC 1.1.1.- such as the gene product ofYMR318C (classified, for example, under EC 1.1.1.2, see GenbankAccession No. CAA90836.1) (Larroy et al., 2002, Biochem J., 361(Pt 1),163-172), cpnD (Iwaki et al., 2002, Appl. Environ. Microbiol.,68(11):5671-5684), or gabD (Lutke-Eversloh & Steinbuchel, 1999, FEMSMicrobiology Letters, 181(1):63-71), or a polypeptide having6-hydroxyhexanoate dehydrogenase activity classified, for example, underEC 1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11):5158-5162); followed by conversion to7-aminoheptanoate by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQID NO: 10), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1,SEQ ID NO: 12). See FIG. 5.

Pathway Using 7-Aminoheptanoate, 7-Hydroxyheptanoate, or PimelateSemialdehyde as Central Precursor to Heptamethylenediamine

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor 7-aminoheptanoate by conversion of 7-aminoheptanoateto 7-aminoheptanal by a polypeptide having carboxylate reductaseactivity classified, for example, under EC 1.2.99.6 such as the geneproduct of car in combination with a phosphopantetheine transferaseenhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt genefrom Nocardia) or the gene products of GriC and GriD from Streptomycesgriseus; followed by conversion of 7-aminoheptanal toheptamethylenediamine by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA811135.1, SEQ ID NO: 11), an Escherichia coli (see Genbank AccessionNo. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see GenbankAccession No. AEA39183.1, SEQ ID NO: 13). See FIG. 6.

The polypeptide having carboxylate reductase activity encoded by thegene product of car and enhancer npt or sfp has broad substratespecificity, including terminal difunctional C4 and C5 carboxylic acids(Venkitasubramanian et al., Enzyme and Microbiol Technology, 2008, 42,130-137).

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor 7-hydroxyheptanoate (which can be produced asdescribed in FIG. 7), by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as from a Mycobacteriummarinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), aMycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ IDNO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1,SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No.ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see GenbankAccession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (seeGenbank Accession No. ADG98140.1, SEQ ID NO: 7), in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp (GenbankAccession No. CAA44858.1, SEQ ID NO:21) gene from Bacillus subtilis ornpt (Genbank Accession No. ABI83656.1, SEQ ID NO:22) gene fromNocardia), or the gene product of GriC & GriD (Suzuki et al., J.Antibiot., 2007, 60(6), 380-387); followed by conversion of7-oxoheptanol to 7-aminoheptanol by a polypeptide having ω-transaminaseactivity classified, for example, under EC 2.6.1.- such as 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from aChromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ IDNO: 8), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1,SEQ ID NO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11); followed by conversion to 7-aminoheptanal bya polypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, orEC 1.1.1.184) such as the gene product of YMR318C (Genbank Accession No.CAA90836.1) or YqhD (from E. coli. GenBank Accession No. AAA69178.1)(Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002,Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol.Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No.CAA81612.1; followed by conversion to heptamethylenediamine classified,for example, under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see Genbank AccessionNo. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see GenbankAccession No. AEA39183.1, SEQ ID NO: 13). See FIG. 6.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor 7-aminoheptanoate by conversion of 7-aminoheptanoateto N7-acetyl-7-aminoheptanoate by a polypeptide havingN-acetyltransferase activity such as a polypeptide having lysineN-acetyltransferase activity classified, for example, under EC 2.3.1.32;followed by conversion to N7-acetyl-7-aminoheptanal by a polypeptidehaving carboxylate reductase activity such as from a Mycobacteriumsmegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), aMycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ IDNO: 6), or a Segniliparus rotundus (see Genbank Accession No.ADG98140.1, SEQ ID NO: 7), in combination with a phosphopantetheinetransferase enhancer (e.g., encoded by a sfp (Genbank Accession No.CAA44858.1, SEQ ID NO:21) gene from Bacillus subtilis or npt (GenbankAccession No. ABI83656.1, SEQ ID NO:22) gene from Nocardia), or the geneproduct of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6),380-387); followed by conversion to N7-acetyl-1,7-diaminoheptane by apolypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as from a Pseudomonas aeruginosa (seeGenbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae(see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), a Rhodobactersphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 11), anEscherichia coli (see Genbank Accession No. AAA57874.1, SEQ ID NO: 12),or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO:13); followed by conversion to heptamethylenediamine by a polypeptidehaving acetylputrescine deacetylase activity classified, for example,under EC 3.5.1.17 or EC 3.5.1.62. See, FIG. 6.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor pimelate semialdehyde by conversion of pimelatesemialdehyde to heptanedial by a polypeptide having carboxylatereductase activity classified, for example, under EC 1.2.99.6 such asfrom a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQID NO: 7), in combination with a phosphopantetheine transferase enhancer(e.g., encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:21)gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1,SEQ ID NO:22) gene from Nocardia), or the gene product of GriC & GriD(Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed byconversion to 7-aminoheptanal by a polypeptide having ω-transaminaseactivity classified, for example, under EC 2.6.1.- such as 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from aChromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ IDNO: 8), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1,SEQ ID NO: 9), a Pseudomonas syringae (see Genbank Accession No.AAY39893.1, SEQ ID NO: 10), a Rhodobacter sphaeroides (see GenbankAccession No. ABA81135.1, SEQ ID NO: 11), an Escherichia coli (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis(see Genbank Accession No. AEA39183.1, SEQ ID NO: 13). See FIG. 6.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor 1,7-heptanediol by conversion of 1,7-heptanediol to7-hydroxyheptanal by a polypeptide having alcohol dehydrogenase activityclassified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2,EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C(Genbank Accession No. CAA90836.1) or YqhD (from E. coli, GenBankAccession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1; followed by conversion of7-oxoheptanal to 7-aminoheptanol by a polypeptide having ω-transaminaseactivity classified, for example, under EC 2.6.1.- such as 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from aChromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ IDNO: 8), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1,SEQ ID NO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11); followed by conversion to 7-aminoheptanal bya polypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, orEC 1.1.1.184) such as the gene product of YMR318C (Genbank Accession No.CAA90836.1) or YqhD) (from E. coli, GenBank Accession No. AAA69178.1)(Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002,Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol.Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No.CAA81612.1; followed by conversion to heptamethylenediamine by apolypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 9), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:10), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1,SEQ ID NO: 11), an Escherichia coli (see Genbank Accession No.AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank AccessionNo. AEA39183.1, SEQ ID NO: 13). See FIG. 6.

Pathways Using 7-Hydroxyheptanoate or Heptanoyl-CoA as Central Precursorto 1,7-Heptanediol

In some embodiments, 1,7 heptanediol is synthesized from the centralprecursor 7-hydroxyheptanoate by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as from a Mycobacteriummarinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), aMycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ IDNO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1,SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No.ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see GenbankAccession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (seeGenbank Accession No. ADG98140.1, SEQ ID NO: 7), in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp (GenbankAccession No. CAA44858.1, SEQ ID NO:21) gene from Bacillus subtilis ornpt (Genbank Accession No. ABI83656.1, SEQ ID NO:22) gene fromNocardia), or the gene product of GriC & GriD (Suzuki et al., J.Antibiot., 2007, 60(6), 380-387); followed by conversion of7-hydroxyheptanal to 1,7 heptanediol by a polypeptide having alcoholdehydrogenase activity classified, for example, under EC 1.1.1.- such asEC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E.coli, GenBank Accession No. AAA69178.1) (see, e.g., Liu et al.,Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J.,361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol.,89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1(from Geobacillus stearothermophilus). See, FIG. 8.

In some embodiments, heptanoyl-CoA can be converted to heptanoic acidheptyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.- (84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 26);followed by conversion to heptanoic acid 7-hydroxy heptyl ester and/or7-hydroxyheptanoic acid 7-hydroxyheptyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (1,3). For example, a polypeptide having monooxygenaseactivity can be, for example, from the CYP153A family, the CYP52A3(Genbank Accession No: BAA00371.1, SEQ ID NO: 29) family or the alkBfamily such as the gene product of alkBGT from Pseudomonas putida(Genbank Accession No. CAB51047.1, SEQ ID NO: 28); followed byconversion of heptanoic acid 7-hydroxy heptyl ester and/or7-hydroxyheptanoic acid 7-hydroxyheptyl to 1,7-heptanediol using apolypeptide having esterase activity classified, for example, under EC3.1.1.- (1,6) such as the gene product of EstC (see GenBank AccessionNo. AAC60471.2, SEQ ID NO: 27). See FIG. 8.

Cultivation Strategy

In some embodiments, the cultivation strategy entails achieving anaerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrificationcultivation condition. Enzymes characterized in vitro as being oxygensensitive require a micro-aerobic cultivation strategy maintaining avery low dissolved oxygen concentration (See, for example, Chayabatra &Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson andBouwer, 1997, Journal of Industrial Microbiology and Biotechnology,18(2-3), 116-130).

In some embodiments, the cultivation strategy entails nutrientlimitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a final electron acceptor other than oxygen such asnitrates can be utilized.

In some embodiments, a cell retention strategy using, for example,ceramic membranes can be employed to achieve and maintain a high celldensity during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C7 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cuprimaidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90:885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin andPrather, J. Biotechnol., 2009, 139:61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Weeet al., Food Technol. Biotechnol., 2006, 44(2): 163-172; Ohashi et al.,J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1):152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C7 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C7 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the only,step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class. In addition,enzymes in a pathway that require a particular co-factor can be replacedwith an enzyme that has similar or identical activity and specificityfor a different co-factor. For example, one or more steps in a pathwaythat use an enzyme with specificity for NADH can be replaced with anenzyme having similar or identical activity and specificity for NADPH.Similarly, one or more steps in a pathway that use an enzyme withspecificity for NADPH can be replaced with an enzyme having similar oridentical activity and specificity for NADH.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined here can begene dosed, i.e., overexpressed, into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C7 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C7 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C7 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemicalnetwork can be attenuated or augmented to (1) ensure the intracellularavailability of acetyl-CoA and propanoyl-CoA, (2) create a NADH or NADPHimbalance that may be balanced via the formation of one or more C7building blocks, (3) prevent degradation of central metabolites, centralprecursors leading to and including one or more C7 building blocksand/or (4) ensure efficient efflux from the cell.

In some embodiments requiring intracellular availability ofpropanoyl-CoA for C7 building block synthesis, endogenous enzymescatalyzing the hydrolysis of propionoyl-CoA and acetyl-CoA such asshort-chain length polypeptides having thioesterase activity can beattenuated in the host organism.

In some embodiments requiring the intracellular availability ofpropanoyl-CoA for C7 building block synthesis, endogenous enzymesconsuming propanoyl-CoA to succinyl-CoA via the methylcitrate cycle suchas a polypeptide having methylcitrate synthase activity can beattenuated in the host organism (Bramer & Steinbuchel, 2001,Microbiology, 147: 2203-2214).

In some embodiments requiring the intracellular availability ofpropanoyl-CoA via L-threonine as central metabolite for C7 buildingblock synthesis, a feedback-resistant polypeptide having threoninedeaminase activity can be genetically engineered into the host organism(Tseng et al., Microbial Cell Factories, 2010, 9:96).

In some embodiments requiring condensation of acetyl-CoA andpropanoyl-CoA for C7 building block synthesis, one or more endogenouspolypeptide having β-ketothiolases activity catalyzing the condensationof only acetyl-CoA to acetoacetyl-CoA such as the endogenous geneproducts of AtoB or phaA can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, an endogenous polypeptidehaving phosphotransacetylase activity generating acetate such as pta canbe attenuated (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, an endogenous gene in anacetate synthesis pathway encoding a polypeptide having acetate kinaseactivity, such as ack, can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of pyruvate to lactatesuch as a polypeptide having lactate dehydrogenase activity encoded byIdhA can be attenuated (Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, endogenous genesencoding enzymes, such as a polypeptide having menaquinol-fiumarateoxidoreductase activity, that catalyze the degradation ofphophoenolpyruvate to succinate such asfrdBC can be attenuated (see,e.g., Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the polypeptide having alcohol dehydrogenase activityencoded by adhE can be attenuated (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C7building block synthesis, a recombinant polypeptide having formatedehydrogenase activity can be overexpressed in the host organism (Shenet al., 2011, supra).

In some embodiments, where pathways require excess NADH or NADPHco-factor for C7 building block synthesis, a polypeptide havingtranshydrogenase activity dissipating the cofactor imbalance can beattenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as polypeptidehaving pyruvate decarboxylase activity can be attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the generation of isobutanol such as a polypeptide having2-oxoaciddecarboxylase activity can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, a recombinant polypeptidehaving acetyl-CoA synthetase activity such as the gene product of acscan be overexpressed in the microorganism (Satoh et al., J. Bioscienceand Bioengineering, 2003, 95(4):335-341).

In some embodiments, carbon flux can be directed into the pentosephosphate cycle to increase the supply of NADPH by attenuating anendogenous polypeptide having glucose-6-phosphate isomerase activity (EC5.3.1.9).

In some embodiments, carbon flux can be redirected into the pentosephosphate cycle to increase the supply of NADPH by overexpression apolypeptide having 6-phosphogluconate dehydrogenase activity and/or apolypeptide having transketolase activity (Lee et al., 2003,Biotechnology Progress, 19(5), 1444-1449).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a gene such as UdhA encoding apolypeptide having puridine nucleotide transhydrogenase activity can beoverexpressed in the host organisms (Brigham et al., Advanced Biofuelsand Bioproducts, 2012, Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 Building Block, a recombinant polypeptide havingglyceraldehyde-3-phosphate-dehydrogenase activity such as GapN can beoverexpressed in the host organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant polypeptide havingmalic enzyme activity such as maeA or maeB can be overexpressed in thehost organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant polypeptide havingglucose-6-phosphate dehydrogenase activity such as zwf can beoverexpressed in the host organisms (Lim et al., J. Bioscience andBioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant polypeptide havingfructose 1,6 diphosphatase activity such as fbp can be overexpressed inthe host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, endogenous polypeptide havingtriose phosphate isomerase activity (EC 5.3.1.1) can be attenuated.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant polypeptide havingglucose dehydrogenase activity such as the gene product of gdh can beoverexpressed in the host organism (Satoh et al., J. Bioscience andBioengineering, 2003, 95(4):335-341).

In some embodiments, endogenous enzymes facilitating the conversion ofNADPH to NADH can be attenuated, such as the NADH generation cycle thatmay be generated via inter-conversion of polypeptide having glutamatedehydrogenases activity classified under EC 1.4.1.2 (NADH-specific) andEC 1.4.1.4 (NADPH-specific).

In some embodiments, an endogenous polypeptide having glutamatedehydrogenase activity (EC 1.4.1.3) that utilizes both NADH and NADPH asco-factors can be attenuated.

In some embodiments, a membrane-bound polypeptide having cytochrome P450activity such as CYP4F3B can be solubilized by only expressing thecytosolic domain and not the N-terminal region that anchors the P450 tothe endoplasmic reticulum (see, for example, Scheller et al., J. Biol.Chem., 1994, 269(17):12779-12783).

In some embodiments, a membrane-bound polypeptide having enoyl-CoAreductase activity can be solubilized via expression as a fusion proteinto a small soluble protein such as a polypeptide having maltose bindingprotein activity (Gloerich et al., FEBS Letters, 2006, 580, 2092-2096).

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, the endogenous polypeptide havingpolyhydroxyalkanoate synthase activity can be attenuated in the hoststrain.

In some embodiments requiring the intracellular availability ofpentanoyl-CoA for C7 building block synthesis, a recombinant polypeptidehaving propionyl-CoA synthetase activity such as the gene product ofPrpE-RS can be overexpressed in the microorganism (Rajashekhara &Watanabe, FEBS Letters, 2004, 556:143-147).

In some embodiments, a polypeptide having L-alanine dehydrogenaseactivity can be overexpressed in the host to regenerate L-alanine frompyruvate as an amino donor for ω-transaminase reactions.

In some embodiments, a polypeptide having L-glutamate dehydrogenaseactivity, a polypeptide having L-glutamine synthetase activity, or apolypeptide having glutamate synthase activity can be overexpressed inthe host to regenerate L-glutamate from 2-oxoglutarate as an amino donorfor ω-transaminase reactions.

In some embodiments, enzymes such as a polypeptide having pimeloyl-CoAdehydrogenase activity classified under, EC 1.3.1.62; a polypeptidehaving acyl-CoA dehydrogenase activity classified, for example, under EC1.3.8.7 or EC 1.3.8.1; and/or a polypeptide having glutaryl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.6 thatdegrade central metabolites and central precursors leading to andincluding C7 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C7 building blocksvia Coenzyme A esterification such as polypeptides having CoA-ligaseactivity (e.g., a pimeloyl-CoA synthetase) classified under, forexample, EC 6.2.1.14 can be attenuated.

In some embodiments, the efflux of a C7 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C7 building block.

The efflux of heptamethylenediamine can be enhanced or amplified byoverexpressing broad substrate range multidrug transporters such as Bitfrom Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem.,272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins &Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA fromStaphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother,38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992.Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 7-aminoheptanoate and heptamethylenediamine can beenhanced or amplified by overexpressing the solute transporters such asthe lysE transporter from Corynebacterium glutamicum (Bellmann et al.,2001, Microbiology, 147, 1765-1774). The efflux of pimelic acid can beenhanced or amplified by overexpressing a dicarboxylate transporter suchas the SucE transporter from Corynebacterium glutamicum (Huhn et al.,Appl. Microbiol. & Biotech., 89(2), 327-335).

Producing C7 Building Blocks Using a Recombinant Host

Typically, one or more C7 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C7 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C7 building block. Once produced, any method can be usedto isolate C7 building blocks. For example, C7 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of pimelic acid and 7-aminoheptanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofheptamethylenediamine and 1,7-heptanediol, distillation may be employedto achieve the desired product purity.

EXAMPLES Example 1 Enzyme Activity of ω-Transaminase Using PimelateSemialdehyde as Substrate and Forming 7-Aminoheptanoate

A nucleotide sequence encoding an N-terminal His-tag was added to thegenes from Chromobacterium violaceum, Pseudomonas syringae, Rhodobactersphaeroides, and Vibrio fluvialis encoding the ω-transaminases of SEQ IDNOs: 8, 10, 11 and 13, respectively (see FIGS. 10F-10H) such thatN-terminal HIS tagged ω-transaminases could be produced. Each of theresulting modified genes was cloned into a pET21a expression vectorunder control of the T7 promoter and each expression vector wastransformed into a BL21[DE3] E. coli host. The resulting recombinant E.coli strains were cultivated at 37° C. in a 250 mL shake flask culturecontaining 50 mL LB media and antibiotic selection pressure, withshaking at 230 rpm. Each culture was induced overnight at 16° C. using 1mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanoateto pimelate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate,10 mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanoate and incubated at 25° C. for 4 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 7-aminoheptanoate demonstrated low baseline conversion of pyruvate to L-alanine. See FIG. 17. The gene productof SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 accepted7-aminoheptanote as substrate as confirmed against the empty vectorcontrol. See FIG. 18.

Enzyme activity in the forward direction (i.e., pimelate semialdehyde to7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO 10,SEQ ID NO 11 and SEQ ID NO 13. Enzyme activity assays were performed ina buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM pimelate semialdehyde, 10 mM L-alanine and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase geneproduct or the empty vector control to the assay buffer containing thepimelate semialdehyde and incubated at 25° C. for 4 h, with shaking at250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene product of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 acceptedpimelate semialdehyde as substrate as confirmed against the empty vectorcontrol. See FIG. 19. The reversibility of the ω-transaminase activitywas confirmed, demonstrating that the ω-transaminases of SEQ ID NO 8,SEQ ID NO 10, SEQ ID NO 11, and SEQ ID NO 13 accepted pimelatesemialdehyde as substrate and synthesized 7-aminoheptanoate as areaction product.

Example 2 Enzyme Activity of Carboxylate Reductase Using Pimelate asSubstrate and Forming Pimelate Semialdehyde

A nucleotide sequence encoding a HIS-tag was added to the genes fromSegniliparus rugosus and Segniliparus rotundus that encode thecarboxylate reductases of SEQ ID NOs: 4 (EFV11917.1) and 7 (ADG98140.1),respectively (see FIG. 10C and FIG. 10F), such that N-terminal HIStagged carboxylate reductases could be produced. Each of the modifiedgenes was cloned into a pET Duet expression vector along with a sfp geneencoding a HIS-tagged phosphopantetheine transferase from Bacillussubtilis, both under the T7 promoter. Each expression vector wastransformed into a BL21[DE3]E. coli host and the resulting recombinantE. coli strains were cultivated at 37° C. in a 250 mL shake flaskculture containing 50 mL LB media and antibiotic selection pressure,with shaking at 230 rpm. Each culture was induced overnight at 37° C.using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation. The carboxylate reductases and phosphopantetheinetransferases were purified from the supernatant using Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated via ultrafiltration.

Enzyme activity assays (i.e., from pimelate to pimelate semialdehyde)were performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate, 10 mMMgCl₂, 1 mM ATP and 1 mM NADPH. Each enzyme activity assay reaction wasinitiated by adding purified carboxylate reductase andphosphopantetheine transferase gene products or the empty vector controlto the assay buffer containing the pimelate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without pimelatedemonstrated low base line consumption of NADPH. See bars for EFV11917.1and ADG98140.1 in FIG. 12.

The gene products of SEQ ID NO: 4 (EFV11917.1) and SEQ ID NO: 7(ADG98140.1), enhanced by the gene product of sfp, accepted pimelate assubstrate, as confirmed against the empty vector control (see FIG. 13),and synthesized pimelate semialdehyde.

Example 3 Enzyme Activity of Carboxylate Reductase Using7-Hydroxyheptanoate as Substrate and Forming 7-Hydroxyheptanal

A nucleotide sequence encoding a His-tag was added to the genes fromMycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus,Mycobacterium smegmatis, Mycobacterium massiliense, and Segniliparusrotundus that encode the carboxylate reductases of SEQ ID NOs: 2-7,respectively (GenBank Accession Nos. ACC40567.1, ABK71854.1, EFV11917.1,ABK75684.1, EIV11143.1, and ADG98140.1, respectively) (see FIGS.10A-10F) such that N-terminal HIS tagged carboxylate reductases could beproduced. Each of the modified genes was cloned into a pET Duetexpression vector alongside a sfp gene encoding a His-taggedphosphopantetheine transferase from Bacillus subtilis, both undercontrol of the T7 promoter. Each expression vector was transformed intoa BL21 [DE3] E. coli host along with the expression vectors from Example3. Each resulting recombinant E. coli strain was cultivated at 37° C. ina 250 mL shake flask culture containing 50 mL LB media and antibioticselection pressure, with shaking at 230 rpm. Each culture was inducedovernight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxyheptanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 7-hydroxyheptanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 7-hydroxyheptanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without7-hydroxyheptanoate demonstrated low base line consumption of NADPH. SeeFIG. 12.

The gene products of SEQ ID NO 2-7, enhanced by the gene product of sfp,accepted 7-hydroxyheptanoate as substrate as confirmed against the emptyvector control (see FIG. 14), and synthesized 7-hydroxyheptanal.

Example 4 Enzyme Activity of ω-Transaminase for 7-Aminoheptanol, Forming7-Oxoheptanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas syringae and Rhodobactersphaeroides genes encoding the ω-transaminases of SEQ ID NOs: 8, 10 and11, respectively (see FIG. 10F and FIG. 10G) such that N-terminal HIStagged ω-transaminases could be produced. The modified genes were clonedinto a pET21a expression vector under the T7 promoter. Each expressionvector was transformed into a BL21[DE3] E. coli host. Each resultingrecombinant E. coli strain were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanolto 7-oxoheptanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 7-aminoheptanol had low base lineconversion of pyruvate to L-alanine. See FIG. 17.

The gene products of SEQ ID NO 8, 10 & 11 accepted 7-aminoheptanol assubstrate as confirmed against the empty vector control (see FIG. 22)and synthesized 7-oxoheptanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID 8, 10 & 11 accept7-oxoheptanol as substrate and form 7-aminoheptanol.

Example 5 Enzyme Activity of ω-Transaminase Using Heptamethylenediamineas Substrate and Forming 7-Aminoheptanal

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the co-transaminases of SEQ ID NOs: 8-13, respectively (seeFIGS. 10F-10H) such that N-terminal HIS tagged ω-transaminases could beproduced. The modified genes were cloned into a pET21a expression vectorunder the T7 promoter. Each expression vector was transformed into aBL21 [DE3] E. coli host. Each resulting recombinant E. coli strain werecultivated at 37° C. in a 250 mL shake flask culture containing 50 mL LBmedia and antibiotic selection pressure, with shaking at 230 rpm. Eachculture was induced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,heptamethylenediamine to 7-aminoheptanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMheptamethylenediamine, 10 mM pyruvate, and 100 M pyridoxyl 5′ phosphate.Each enzyme activity assay reaction was initiated by adding cell freeextract of the ω-transaminase gene product or the empty vector controlto the assay buffer containing the heptamethylenediamine and thenincubated at 25° C. for 4 h, with shaking at 250 rpm. The formation ofL-alanine was quantified via RP-HPLC.

Each enzyme only control without heptamethylenediamine had low base lineconversion of pyruvate to L-alanine. See FIG. 17.

The gene products of SEQ ID NO 8-13 accepted heptamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 20)and synthesized 7-aminoheptanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID 8-13 accept 7-aminoheptanalas substrate and form heptamethylenediamine.

Example 6 Enzyme Activity of Carboxylate Reductase forN7-Acetyl-7-Aminoheptanoate, Forming N7-Acetyl-7-Aminoheptanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 3, 6, and 7 (see Examples 2 and 3, and FIGS. 10B, 10E,and 10F) for converting N7-acetyl-7-aminoheptanoate toN7-acetyl-7-aminoheptanal was assayed in triplicate in a buffer composedof a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mMN7-acetyl-7-aminoheptanoate, 10 mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Theassays were initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the N7-acetyl-7-aminoheptanoate then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control withoutN7-acetyl-7-aminoheptanoate demonstrated low base line consumption ofNADPH. See FIG. 12.

The gene products of SEQ ID NO 3, 6, and 7, enhanced by the gene productof sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmedagainst the empty vector control (see FIG. 15), and synthesizedN7-acetyl-7-aminoheptanal.

Example 7 Enzyme Activity of ω-Transaminase UsingN7-Acetyl-1,7-Diaminoheptane, and Forming N7-Acetyl-7-Aminoheptanal

The activity of the N-terminal His-tagged (ω-transaminases of SEQ IDNOs: 8-13 (see Example 5, and FIGS. 10F-10H) for convertingN7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN7-acetyl-1,7-diaminoheptane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N7-acetyl-1,7-diaminoheptanedemonstrated low base line conversion of pyruvate to L-alanine. See FIG.17.

The gene product of SEQ ID NOs: 8-13 acceptedN7-acetyl-1,7-diaminoheptane as substrate as confirmed against the emptyvector control (see FIG. 21) and synthesized N7-acetyl-7-aminoheptanalas reaction product.

Given the reversibility of the ω-transaminase activity (see Example 1),the gene products of SEQ ID NOs: 8-13 accept N7-acetyl-7-aminoheptanalas substrate forming N7-acetyl-1,7-diaminoheptane.

Example 8 Enzyme Activity of Carboxylate Reductase Using PimelateSemialdehyde as Substrate and Forming Heptanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (seeExample 3 and FIG. 10F) was assayed using pimelate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM pimelate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the pimelate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout pimelate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 12.

The gene product of SEQ ID NO 7, enhanced by the gene product of sfp,accepted pimelate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 16) and synthesized heptanedial.

Example 9 Enzyme Activity of CYP153 Monooxygenase Using Heptanoate asSubstrate in Forming 7-Hydroxyheptanoate

A nucleotide sequence encoding a HIS tag was added to the Polaromonassp. JS666, Mycobacterium sp. HXN-1500 and Mycobacterium austroafricanumgenes respectively encoding (1) the monooxygenases (SEQ ID NOs: 14-16),(2) the associated ferredoxin reductase partner (SEQ ID NOs: 17-18) andthe specie's ferredoxin (SEQ ID NOs: 19-20). For the Mycobacteriumaustroafricanum monooxygenase, Mycobacterium sp. HXN-1500 oxidoreductaseand ferredoxin partners were used. The three modified protein partnerswere cloned into a pgBlue expression vector under a hybrid pTacpromoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 500 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure. Each culture was induced for 24 h at 28°C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and the cells made permeableusing Y-Per™ solution (ThermoScientific, Rockford, Ill.) at roomtemperature for 20 min. The permeabilized cells were held at 0° C. inthe Y-Per™ solution.

Enzyme activity assays were performed in a buffer composed of a finalconcentration of 25 mM potassium phosphate buffer (pH=7.8), 1.7 mMMgSO₄, 2.5 mM NADPH and 30 mM heptanoate. Each enzyme activity assayreaction was initiated by adding a fixed mass of wet cell weight ofpermeabilized cells suspended in the Y-Per™ solution to the assay buffercontaining the heptanoate and then incubated at 28° C. for 24 h, withshaking at 1400 rpm in a heating block shaker. The formation of7-hydroxyheptanoate was quantified via LC-MS.

The monooxygenase gene products of SEQ ID NO 14-16 along with reductaseand ferredoxin partners, accepted heptanoate as substrate as confirmedagainst the empty vector control (see FIG. 11) and synthesized7-hydroxyheptanoate as reaction product.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-83. (canceled)
 84. A method of producing 7-hydroxyheptanoate methylester in a recombinant host via fermentation, said method comprising:enzymatically converting heptanoate to heptanoate methyl ester using apolypeptide having fatty acid O-methyltransferase activity; andenzymatically converting heptanoate methyl ester to 7-hydroxyheptanoatemethyl ester using a polypeptide having monooxygenase activity.
 85. Themethod of claim 84, wherein: said polypeptide having fatty acidO-methyltransferase activity has at least 70% sequence identity to anamino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 24, or SEQ IDNO: 25; said polypeptide having monooxygenase activity has at least 70%sequence identity to an amino acid sequence set forth in SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 28, and/or SEQ ID NO:
 29. 86.The method of claim 84, wherein heptanoate is enzymatically producedfrom heptanoyl-CoA using: a polypeptide having thioesterase activity; ora polypeptide having butanal dehydrogenase activity and a polypeptidehaving aldehyde dehydrogenase activity.
 87. The method of claim 84,wherein heptanoate is enzymatically produced from heptanoyl-CoA using apolypeptide having thioesterase activity, wherein said polypeptidehaving thioesterase activity has at least 70% sequence identity to theamino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 33, or SEQ IDNO:
 34. 88. The method of claim 86, wherein heptanoyl-CoA is producedfrom acetyl-CoA and propanoyl-CoA via two cycles of CoA-dependent carbonchain elongation, wherein each of said two cycles of CoA-dependentcarbon chain elongation comprises using: a polypeptide havingβ-ketothiolase activity or a polypeptide having acetyl-CoA carboxylaseactivity and a polypeptide having β-ketoacyl-[acp] synthase activity; apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity or apolypeptide having 3-oxoacyl-CoA reductase activity; a polypeptidehaving enoyl-CoA hydratase activity; and a polypeptide havingtrans-2-enoyl-CoA reductase activity.
 89. The method of claim 84, saidmethod further comprising enzymatically converting 7-hydroxyheptanoatemethyl ester to 7-hydroxyheptanoate using a polypeptide havingdemethylase activity or a polypeptide having esterase activity.
 90. Themethod of claim 89, said method further comprising enzymaticallyconverting 7-hydroxyheptanoate to at least one product chosen frompimelic acid, pimelate semialdehyde, 7-aminoheptanoate,heptamethylenediamine, and 1,7-heptanediol.
 91. The method of claim 89,said method further comprising enzymatically converting7-hydroxyheptanoate to pimelate semialdehyde using a polypeptide havingalcohol dehydrogenase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, or a polypeptide having monooxygenase activity.92. The method of claim 89, said method further comprising:enzymatically converting 7-hydroxyheptanoate to 1,7-heptanediol using apolypeptide having carboxylate reductase activity and a polypeptidehaving alcohol dehydrogenase activity, wherein said polypeptide havingcarboxylate reductase is optionally enhanced by gene product of sfp; andoptionally further comprising enzymatically converting 1,7-heptanediolto heptamethylenediamine using a polypeptide having alcoholdehydrogenase activity and a polypeptide having ω-transaminase activity.93. The method of claim 91, said method further comprising:enzymatically converting pimelate semialdehyde to pimelic acid using apolypeptide having 5-oxopentanoate dehydrogenase activity, a polypeptidehaving 6-oxohexanoate dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having monooxygenase activity;enzymatically converting pimelate semialdehyde to 7-aminoheptanoateusing a polypeptide having ω-transaminase activity; or enzymaticallyconverting pimelate semialdehyde to heptamethylenediamine using at leastone polypeptide chosen from a polypeptide having carboxylate reductaseactivity and a polypeptide having ω-transaminase activity, wherein saidpolypeptide having carboxylate reductase is optionally enhanced by geneproduct of sfp.
 94. The method of claim 93, said method comprisingenzymatically converting pimelate semialdehyde to 7-aminoheptanoateusing a polypeptide having ω-transaminase activity, and furthercomprising enzymatically converting 7-aminoheptanoate toheptamethylenediamine using: at least one polypeptide chosen from apolypeptide having carboxylate reductase activity and a polypeptidehaving ω-transaminase activity, wherein the polypeptide havingcarboxylate reductase is optionally enhanced by gene product of sfp; andoptionally a polypeptide having deacetylase activity or a polypeptidehaving acetylputrescine deacetylase activity.
 95. The method of claim84, wherein said recombinant host is: subjected to a cultivationstrategy under aerobic, anaerobic or, micro-aerobic cultivationconditions; cultured under conditions of nutrient limitation; and/orretained using a ceramic membrane to maintain a high cell density duringfermentation.
 96. The method of claim 84, wherein the principal carbonsource fed to the recombinant host is a biological feedstock ornon-biological feedstock.
 97. The method of claim 84, wherein: saidbiological feedstock is or derives from monosaccharides, disaccharides,lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formicacid, triglycerides, glycerol, fatty acids, agricultural waste,condensed distillers' solubles, or municipal waste; or saidnon-biological feedstock is or derives from natural gas, syngas, CO₂/H₂,methanol, ethanol, benzoate, non-volatile residue (NVR) caustic washwaste stream from cyclohexane oxidation processes, or terephthalicacid/isophthalic acid mixture waste streams.
 98. The method of claim 84,wherein said recombinant host is: a prokaryote chosen from the generaEscherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas,Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus; or aeukaryote chosen from the genera Aspergillus, Saccharomyces, Pichia,Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces.
 99. Themethod of claim 84, wherein said recombinant host is: a prokaryotechosen from Escherichia coli, Clostridium ljungdahlii, Clostridiumautoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum,Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens,Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans,Bacillus subtilis, Lactobacillus delbrueckii, Lactococcus lactis, andRhodococcus equi; or a eukaryote chosen from Aspergillus niger,Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica,Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans,and Kluyveromyces lactis.
 100. The method of claim 84, wherein saidrecombinant host: comprises at least one attenuated enzyme chosen from:a polypeptide having polyhydroxyalkanoate synthase activity; apolypeptide having acetyl-CoA thioesterase activity; a polypeptidehaving propanoyl-CoA thioesterase activity; a polypeptide havingphosphotransacetylase forming acetate activity; a polypeptide havingacetate kinase activity; a polypeptide having lactate dehydrogenaseactivity; a polypeptide having menaquinol-fumarate oxidoreductaseactivity; a polypeptide having 2-oxoacid decarboxylase activityproducing isobutanol; a polypeptide having alcohol dehydrogenaseactivity forming ethanol; a polypeptide having triose phosphateisomerase activity; a polypeptide having pyruvate decarboxylaseactivity; a polypeptide having glucose-6-phosphate isomerase activity; apolypeptide having transhydrogenase activity dissipating the cofactorgradient; a polypeptide having glutamate dehydrogenase activity specificfor the cofactor used to create a gradient; a NADH/NADPH-utilizingpolypeptide having glutamate dehydrogenase activity; a polypeptidehaving pimeloyl-CoA dehydrogenase activity; a polypeptide havingacyl-CoA dehydrogenase activity accepting C7 building blocks and centralprecursors as substrates; a polypeptide having glutaryl-CoAdehydrogenase activity; and a polypeptide having pimeloyl-CoA synthetaseactivity; and/or overexpresses at least one gene encoding a polypeptidechosen from: a polypeptide having acetyl-CoA synthetase activity; apolypeptide having propionate-CoA synthetase activity; a polypeptidehaving 6-phosphogluconate dehydrogenase activity; a polypeptide havingtransketolase activity; a polypeptide having puridine nucleotidetranshydrogenase activity; a polypeptide having formate dehydrogenaseactivity; a polypeptide having glyceraldehyde-3P-dehydrogenase activity;a polypeptide having malic enzyme activity; a polypeptide havingglucose-6-phosphate dehydrogenase activity; a polypeptide havingfructose 1,6 diphosphatase activity; a polypeptide having L-alaninedehydrogenase activity; a polypeptide having L-glutamate dehydrogenaseactivity; a polypeptide having L-glutamine synthetase activity; apolypeptide having lysine transporter activity; a polypeptide havingdicarboxylate transporter activity; and a polypeptide having multidrugtransporter activity.
 101. A recombinant host comprising at least oneexogenous nucleic acid encoding a polypeptide having monooxygenaseactivity and at least one nucleic acid encoding a polypeptide havingfatty acid O-methyltransferase activity, wherein: said polypeptidehaving monooxygenase activity has at least 70% sequence identity to anamino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 28, or SEQ ID NO: 29; and said polypeptide havingfatty acid O-methyltransferase activity has at least 70% sequenceidentity to an amino acid sequence set forth in SEQ ID NO: 23, SEQ IDNO: 24, or SEQ ID NO: 25, and said recombinant host produces7-hydroxyheptanoate methyl ester.
 102. The recombinant host of claim101, said recombinant host further comprising an exogenous polypeptidehaving demethylase activity classified under EC 2.1.1.- or a polypeptidehaving esterase activity classified under EC 3.1.1.-, and said hostfurther produces 7-hydroxyheptanoate.
 103. The recombinant host of claim102, said recombinant host further comprising at least one exogenouspolypeptide chosen from: a polypeptide having 4-hydroxybutyratedehydrogenase activity; a polypeptide having 5-hydroxypentanoatedehydrogenase activity; a polypeptide having 5-oxopentanoatedehydrogenase activity; a polypeptide having 6-hydroxyhexanoatedehydrogenase activity; a polypeptide having 6-oxohexanoatedehydrogenase activity; a polypeptide having 7-oxoheptanoatedehydrogenase activity; a polypeptide having acetylputrescinedeacetylase activity; a polypeptide having alcohol dehydrogenaseactivity; a polypeptide having aldehyde dehydrogenase activity; apolypeptide having carboxylate reductase activity; a polypeptide havingdeacetylase activity; and a polypeptide having ω-transaminase activity.104. The recombinant host of claim 102, said recombinant host furthercomprising: an exogenous polypeptide having β-ketothiolase activity oran exogenous polypeptide having acetyl-CoA carboxylase activity and anexogenous polypeptide having β-ketoacyl-[acp] synthase activity; anexogenous polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity oran exogenous polypeptide having 3-oxoacyl-CoA reductase activity; anexogenous polypeptide having enoyl-CoA hydratase activity; and anexogenous polypeptide having trans-2-enoyl-CoA reductase activity. 105.The recombinant host of claim 102, said recombinant host furthercomprising: an exogenous polypeptide having thioesterase activity; or anexogenous polypeptide having butanal dehydrogenase activity and anexogenous polypeptide having aldehyde dehydrogenase activity.
 106. Amethod of producing a bio-based polymer or resin comprising at least onebio-derived seven-carbon compound chosen from pimelic acid, pimelatesemialdehyde, 7-aminoheptanoate acid, 7-hydroxyheptanoate,heptamethylenediamine, and 1,7-heptanediol, said method comprising:producing said at least one bio-derived seven-carbon compound using themethod of claim 90; and chemically reacting said at least onebio-derived seven-carbon compound with itself or another compound in apolymer-producing reaction or resin-producing reaction.
 107. A method ofproducing a molded product comprising producing a polymer or resin usingthe method of claim 106 and forming said polymer or resin into a moldedproduct.
 108. A bio-derived product, bio-based product, orfermentation-derived product, wherein said product comprises: (i) atleast one bio-derived, bio-based, or fermentation-derived compoundproduced using the method of claim 90 or a composition comprising saidat least one bio-derived, bio-based, or fermentation-derived compound;(ii) a bio-derived, bio-based, or fermentation-derived polymercomprising the bio-derived, bio-based, or fermentation-derivedcomposition or compound of (i), or any combination thereof; (iii) abio-derived, bio-based, or fermentation-derived resin comprising thebio-derived, bio-based, or fermentation-derived compound or compositionof (i) or any combination thereof, or the bio-derived, bio-based, orfermentation-derived polymer of (ii), or any combination thereof; (iv) amolded substance obtainable by molding the bio-derived, bio-based, orfermentation-derived polymer of (ii) or the bio-derived, bio-based, orfermentation-derived of (iii), or any combination thereof; (v) abio-derived, bio-based, or fermentation-derived formulation comprisingthe bio-derived, bio-based, or fermentation-derived composition orcompound of (i), the bio-derived, bio-based, or fermentation-derivedpolymer of (ii), the bio-derived, bio-based, or fermentation-derivedresin of (iii), the bio-derived, bio-based, or fermentation-derivedmolded substance of (iv), or any combination thereof; or (vi) abio-derived, bio-based, or fermentation-derived semi-solid or anon-semi-solid stream, comprising the bio-derived, bio-based, orfermentation-derived compound or composition of (i), the bio-derived,bio-based, or fermentation-derived polymer of (ii), the bio-derived,bio-based, or fermentation-derived resin of (iii), the bio-derived,bio-based, or fermentation-derived formulation of (v), the bio-derived,bio-based, or fermentation-derived molded substance of (iv), or anycombination thereof.